Treatment of cancer with immune stimulators

ABSTRACT

The present invention provides compositions and methods for treating cancer or a metastasis thereof in a subject. In some embodiments, the methods involve administering a composition comprising therapeutically effective amount of at least one immune stimulator to the subject. In some embodiments, a combination of at least two immune stimulators is used for the treatment. In some embodiments, the combination includes an alpha thymosin peptide and an additional immune stimulator, and/or optionally one or more additional anti-cancer agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and the benefit of U.S. ProvisionalPatent Application Ser. No. 62/066,862, filed on Oct. 21, 2014 and U.S.Provisional Patent Application Ser. No. 62/215,433, filed on Sep. 8,2015, each of which is incorporated by reference for all purposes.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for treatingcancer, such as melanoma, or metastases thereof.

BACKGROUND OF THE INVENTION

Many drugs or drug candidates have been developed for the treatment ofvarious cancers, including some small molecule compounds. However,current treatments for many cancers are not very effective in patientswith specific subsets of cancers, or are too toxic in such patients orin general.

Skin cancer is the most common form of cancer in the United States. In2007, The American Cancer Society estimates that approximately 8,110deaths will occur from melanoma and another 59,940 cases of melanoma areexpected to be diagnosed in this country.

Melanoma is a malignant tumor of melanocytes which are foundpredominantly in skin but also in bowel and the eye (uveal melanoma). Itis one of the rarer types of skin cancer but causes the majority of skincancer related deaths.

The currently available treatment includes surgical removal of thetumor; adjuvant treatment; chemo- and immunotherapy, or radiationtherapy. Of particular danger are metastases of the primary melanomatumor. However, there remains a need in the art for improved treatmentsof melanoma.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods of treating acancer or combination of cancers in a subject. In some embodiments, thesubject is a mammal. In some embodiments, the mammal is a human.

In some embodiments, the methods comprise administering a compositioncomprising therapeutically effective amount of a first immune stimulatorand a second immune stimulator to the subject. In some embodiments, thefirst immune stimulator is an alpha thymosin peptide. In someembodiments, the second immune stimulator is a compound other than IL-2,interferon-α, or IRX-2. In some embodiments, the second immunestimulator comprises a specific immunostimulant. In some embodiment, thesecond immune stimulator comprises a non-specific immunostimulant.

In some embodiment, the second immune stimulator is an immunostimulantthat is effective in treating sepsis. In some embodiment, theimmunostimulant comprises granulocyte macrophage colony stimulatingfactor (GM-CSF), programmed cell death-1 (PD-1) inhibitors and/orinterleukin-7 (IL-7).

In some embodiment, the composition further comprises an additionalanti-cancer agent. In some embodiments, the additional anti-cancer agentis a chemotherapeutic agent.

In some embodiment, the second immune stimulator is administered to saidsubject at a dosage of about 0.01-1000 mg/day.

In some embodiment, the second immune stimulator comprises GM-CSF, andthe dosage of GM-CSF is about 10 to 500 mcg/m², such as about 125 toabout 250 10 to 500 mcg/m².

In some embodiment, the second immune stimulator comprises a PD-1inhibitor. In some embodiments, the PD-1 inhibitor is an agent thatinhibits PD-1, such as an antibody against PD-1. In some embodiments,the PD-1 inhibitor is an agent that inhibits the ligand for PD-1, suchas an antibody against the ligand for PD-1. In some embodiment, thedosage of the PD-1 inhibitor is about 0.1 to 10 mg/kg, such as about 1-5mg/kg, or about 2-3 mg/kg. In some embodiments, the PD-1 inhibitor is ananti-PD-L1 antibody, and the dosage is about 15-20 mg/kg. In someembodiments, the anti-PD-L1 antibody is used at a 1200 mg flat doseevery two, three, or four weeks.

In some embodiments, the second immune stimulator comprises aninterleukin that is not IL-2. In some embodiments, the interleukin isIL-7. In some embodiment, the dosage of IL-7 is about 0.1 to 100 mcg/kg,such as about 1 to 50 mcg/kg, or about 3 to 30 mcg/kg.

In some embodiments, the alpha thymosin peptide is administered to thesubject during at least a portion of the treatment at a dosage within arange of about 0.1 to 100 mg/day, such as about 0.5-50 mg/day, or about0.1-10 mg/day.

In some embodiments, the alpha thymosin peptide is thymosin alpha 1(TA1).

In some embodiments, the methods comprise administration of TA1 dailyfor a period of about 1-10 days, followed by about 1-5 days ofnon-administration of TA1. In some embodiments, TA1 is administereddaily for about 3-5 days, followed by about 2-4 days ofnon-administration of TA1. In some embodiments, TA1 is administereddaily for about 4 days, followed by about 3 day's non-administration ofTA1.

In some embodiments, the methods further comprise administering a kinaseinhibitor. In some embodiments, the kinase inhibitor comprisessorafenib. In some embodiments, the kinase inhibitor is administered tosaid patient at a dosage within a range of about 10-200 mg/kg/day.

In some embodiments, the methods further comprise administering anantineoplastic heat shock apoptosis activator (HSAA). In someembodiments, the HSAA comprises STA-4783 (elesclomol). In someembodiments, the HSAA is administered to said patient at a dosage withina range of about 0.01-100 mg/kg/day.

In some embodiments, the methods further comprise administering aninhibitor of cytotoxic T lymphocyte-associated antigen 4 (CTLA4), suchas an antibody against CTLA4. In some embodiments, the CTLA4 antibodycomprises 9H10, MDC010, 1F4, BNI3, Q01, A01, M08, 1B8, WKH203, ab9984,ab13486, ipilimumab, ticilimumab or a combination thereof. In someembodiments, the CTLA4 antibody is administered to said patient at adosage within a range of about 0.001-50 mg/kg/day.

In some embodiments, the methods further comprise administering analkylating antineoplastic agent (AlkAA). In some embodiments, thealkylating antineoplastic agent (AlkAA) comprises dacarbazine (DTIC). Insome embodiments, the alkylating antineoplastic agent (AlkAA) isadministered to said patient at a dosage within a range of about700-1300 mg/kg/day.

In some embodiments, the methods further comprise administering achemotherapeutic agent to the subject. In some embodiments, thechemotherapeutic agent is dacarbazine (DTIC) or cisplatin.

In some embodiments, the cancer is melanoma.

The present invention also provides methods of treating cancer or ametastasis thereof in a subject comprising administering a compositioncomprising therapeutically effective amount of an immune stimulator,wherein the immune stimulator is effective in treating sepsis. In someembodiments, the cancer is melanoma. In some embodiments, the subject isa mammal. In some embodiments, the mammal is a human. In someembodiments, the immunostimulant that is effective in treating sepsiscomprises granulocyte macrophage colony stimulating factor (GM-CSF),programmed cell death-1 (PD-1) inhibitors and/or interleukin-7 (IL-7),or any combination thereof.

In some embodiments, the composition further comprises an additionalanti-cancer agent. In some embodiments, the additional anti-cancer agentis an alpha thymosin peptide. In some embodiments, the alpha thymosinpeptide is thymosin alpha 1 (TA1).

In some embodiments, the method further comprises administering achemotherapeutic agent to the subject. In some embodiments, thechemotherapeutic agent is dacarbazine (DTIC) or cisplatin.

The present invention also provides methods for determining theresponsiveness of a human subject to cancer treatment. In someembodiments, the cancer is melanoma. In some embodiments, the methodscomprise determining the level of activity of one or more biomarkers ina biological sample from a human subject. In some embodiments, thebiomarkers are selected from the group consisting of IL-1β, IL-4, IL-6,and IL-10. In some embodiments, the cancer treatment is according to themethods described herein.

In some embodiments, a higher than normal level of IL-1β activity isindicative that the human subject is responsive to the treatment.

In some embodiments, a lower than normal level of IL-4 activity isindicative that the human subject is responsive to the treatment.

In some embodiments, a higher than normal level of IL-6 activity isindicative that the human subject is responsive to the treatment.

In some embodiments, a higher than normal level of IL-10 activity isindicative that the human subject is responsive to the treatment.

The present invention also provides methods for determining dosage orregimen for the treatment of cancer in a human subject. In someembodiments, the cancer is melanoma. In some embodiments, the methodscomprise determining the level of activity of one or more biomarkers ina biological sample from a human subject being treated. In someembodiments, the biomarkers are selected from the group consisting ofIL-1β, IL-4, IL-6, and IL-10.

In some embodiments, a decreased level of IL-1β activity after thetreatment is indicative that the treatment is effective. In someembodiments, an unchanged or increased level of IL-1β activity after thetreatment is indicative that the treatment is not effective. The dosageor regimen of the treatment can be modified accordingly.

In some embodiments, an increased level of IL-4 activity after thetreatment is indicative that the human subject is responsive to thetreatment. In some embodiments, an unchanged or decreased level of IL-4activity after the treatment is indicative that the treatment is noteffective. The dosage or regimen of the treatment can be modifiedaccordingly.

In some embodiments, a decreased level of IL-6 activity after thetreatment is indicative that the treatment is effective. In someembodiments, an unchanged or increased level of IL-6 activity after thetreatment is indicative that the treatment is not effective. The dosageor regimen of the treatment can be modified accordingly.

In some embodiments, a decreased level of IL-10 activity after thetreatment is indicative that the treatment is effective. In someembodiments, an unchanged or increased level of IL-10 activity after thetreatment is indicative that the treatment is not effective. The dosageor regimen of the treatment can be modified accordingly.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts antitumor effect of cisplatin in treatment of B16F10murine melanoma model as indicated by tumor volume post tumorinoculation.

FIG. 1B depicts body weight changes of mice in B16F10 murine melanomamodel post tumor inoculation treated with either vehicle or cisplatin.

FIG. 2 depicts antitumor activity of thymosin. Animals with B16F10derived tumor were treated with ZADAXIN™ (thymalfasin). At all dosestested, animals exhibited reduced tumor growth compared with vehicletreated group.

FIG. 3 depicts antitumor activity of thymosin at several differentdoses. At all tested doses, thymosin provided statistically significantreduced tumor growth compared with vehicle treated group.

FIG. 4A depicts evaluation of IL-1β in subcutaneous B16F10 murinemelanoma model in C57BL/6 mice treated with thymosin. ZADAXIN™(thymalfasin) was administered to mice subcutaneously twice a day for 6days at 0.02, 0.06, 0.2, 0.3, 2, or 6 mg/kg 10 μL/g. IL-1β levels werelower in ZADAXIN™ (thymalfasin) treated groups compared to vehicletreated groups at D4 and D7 after start of dosing.

FIG. 4B depicts evaluation of IL-4 in subcutaneous B16F10 murinemelanoma model in C57BL/6 mice treated with thymosin. ZADAXIN™(thymalfasin) was administered to mice subcutaneously twice a day for 6days at 0.02, 0.06, 0.2, 0.3, 2, or 6 mg/kg 10 μL/g. IL-4 levels werehigher in ZADAXIN™ (thymalfasin) treated groups compared to vehicletreated groups at D4 and D7 after start of dosing.

FIG. 4C depicts evaluation of IL-6 in subcutaneous B16F10 murinemelanoma model in C57BL/6 mice treated with thymosin. ZADAXIN™(thymalfasin) was administered to mice subcutaneously twice a day for 6days at 0.02, 0.06, 0.2, 0.3, 2, or 6 mg/kg 10 μL/g. IL-6 levels werelower in ZADAXIN™ (thymalfasin) treated groups compared to vehicletreated groups at D4 and D7 after start of dosing.

FIG. 4D depicts evaluation of IL-10 in subcutaneous B16F10 murinemelanoma model in C57BL/6 mice treated with thymosin. ZADAXIN™(thymalfasin) was administered to mice subcutaneously twice a day for 6days at 0.02, 0.06, 0.2, 0.3, 2, or 6 mg/kg 10 μL/g. IL-10 levels werelower in ZADAXIN™ (thymalfasin) treated groups compared to vehicletreated groups at D4 and D7 after start of dosing.

FIG. 4E depicts evaluation of IFN-gamma in systemic B16F10 murinemelanoma model in C57BL/6 mice with different treatments.

FIG. 4F depicts evaluation of IL-1β in systemic B16F10 murine melanomamodel in C57BL/6 mice with different treatments.

FIG. 4G depicts evaluation of IL-4 in systemic B16F10 murine melanomamodel in C57BL/6 mice with different treatments.

FIG. 4H depicts evaluation of IL-5 in systemic B16F10 murine melanomamodel in C57BL/6 mice with different treatments.

FIG. 4I depicts evaluation of IL-6 in systemic B16F10 murine melanomamodel in C57BL/6 mice with different treatments.

FIG. 4J depicts evaluation of IL-10 in systemic B16F10 murine melanomamodel in C57BL/6 mice with different treatments.

FIG. 4K depicts evaluation of IL-12 in systemic B16F10 murine melanomamodel in C57BL/6 mice with different treatments.

FIG. 4L depicts evaluation of TNF-α in systemic B16F10 murine melanomamodel in C57BL/6 mice with different treatments.

FIG. 5 depicts B16F10 mouse lung metastatic melanoma model and theexperiment design. B16F10 melanoma cells were inoculated into mice atday 0.

FIG. 6A depicts group distribution of lung metastases on day 16 in micetreated with vehicle, thymosin alone, anti-PD-1 alone, thymosin plusanti-PD-1, or cyclophosphamide.

FIG. 6B depicts percent group mean body weight changes from Day 1 inB16MET mice treated with vehicle, thymosin alone, anti-PD-1 alone,thymosin plus anti-PD-1, or cyclophosphamide.

FIG. 7A depicts group distribution of lung metastases on day 15 in micetreated with vehicle, thymosin alone, anti-PD-1 alone, thymosin plusanti-PD-1, or cyclophosphamide at different doses.

FIG. 7B depicts IL-1α in B16F10 mouse lung metastatic melanoma modelafter treatment with vehicle, thymosin alone, anti-PD-1 alone, thymosinplus anti-PD-1, or cyclophosphamide at different doses.

FIG. 7C depicts percent group mean body weight changes from Day 1 inB16F10 mice treated with vehicle, thymosin alone, anti-PD-1 alone,thymosin plus anti-PD-1, or cyclophosphamide.

FIG. 8 depicts lung metastasis foci in the different groups at Day 13post tumor inoculation treated with vehicle, thymosin alone, anti-PD-1alone, thymosin plus anti-PD-1, or cyclophosphamide.

DETAILED DESCRIPTION

The present invention is directed to methods of treating and/orpreventing cancer in a subject. In some embodiments, the cancer ismelanoma or metastases thereof. In some embodiments, the methods involveadministering a composition comprising at least one immune stimulator tothe subject.

In some embodiments, methods of the present invention can be applied inthe treatment of early stage cancers including early neoplasias that maybe small, slow growing, localized and/or nonaggressive, for example,with the intent of curing the disease or causing regression of thecancer, as well as in the treatment of intermediate stage and in thetreatment of late stage cancers including advanced and/or metastaticand/or aggressive neoplasias, for example, to slow the progression ofthe disease, to reduce metastasis or to increase the survival of thepatient. Similarly, the combinations may be used in the treatment of lowgrade cancers, intermediate grade cancers and or high grade cancers.

In some embodiments, methods of the present invention can also be usedin the treatment of indolent cancers, recurrent cancers includinglocally recurrent, distantly recurrent and/or refractory cancers (i.e.cancers that have not responded to treatment), metastatic cancers,locally advanced cancers and aggressive cancers. Thus, an “advanced”cancer includes locally advanced cancer and metastatic cancer and refersto overt disease in a patient, wherein such overt disease is notamenable to cure by local modalities of treatment, such as surgery orradiotherapy. The term “metastatic cancer” refers to cancer that hasspread from one part of the body to another. Advanced cancers may alsobe unresectable, that is, they have spread to surrounding tissue andcannot be surgically removed.

In some embodiments, methods of the present invention can also be usedin the treatment of drug resistant cancers, including multidrugresistant tumors. As is known in the art, the resistance of cancer cellsto chemotherapy is one of the central problems in the management ofcancer.

One skilled in the art will appreciate that many of these categories mayoverlap, for example, aggressive cancers are typically also metastatic.“Aggressive cancer,” as used herein, refers to a rapidly growing cancer.One skilled in the art will appreciate that for some cancers, such asbreast cancer or prostate cancer the term “aggressive cancer” will referto an advanced cancer that has relapsed within approximately the earliertwo-thirds of the spectrum of relapse times for a given cancer, whereasfor other types of cancer, nearly all cases present rapidly growingcancers which are considered to be aggressive. The term can thus cover asubsection of a certain cancer type or it may encompass all of othercancer types.

In some embodiments, cancers to be treated by the methods of the presentinvention in include, but are not limited to, AIDS-related cancers,adrenocortical cancer, anal cancer, bladder cancer, bowel cancer, brainand central nervous system cancers, breast cancer, carcinoid cancers,cervical cancer, chondrosarcoma, choriocarcinoma, colorectal cancer,endocrine cancers, endometrial cancer, Ewing's sarcoma, eye cancer,gastric cancer, gastrointestinal cancer, genitourinary cancers, glioma,gynecological cancer, head and neck cancer, hepatocellular cancer,Hodgkin's disease, hypopharyngeal cancer, islet cell cancer, Kaposi'ssarcoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lungcancer (e.g., Non-Small Cell Lung Cancer), lymphoma, melanoma, basalcell carcinoma, mesothelioma, myeloma, nasopharyngeal cancer,neuroblastoma, non-Hodgkin's lymphoma, esophagael cancer, osteosarcoma,ovarian cancer, pancreatic cancer, pituitary cancer, renal cellcarcinoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, sarcoma,skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer,thymus cancer, thyroid cancer, transitional cell cancer, trophoblasticcancer, uterine cancer, vaginal cancer, Waldenstrom's macroglobulinemia,Wilm's cancer, and leukemia.

According to the methods of the present invention, the term “subject,”and variants thereof as used herein, includes any subject that has, issuspected of having, or is at risk for having a disease or condition.Suitable subjects (or patients) include mammals, such as laboratoryanimals (e.g., mouse, rat, rabbit, guinea pig), farm animals, anddomestic animals or pets (e.g., cat, dog). Non-human primates and,preferably, human patients, are included. A subject “at risk” may or maynot have detectable disease, and may or may not have displayeddetectable disease prior to the diagnostic or treatment methodsdescribed herein. “At risk” denotes that a subject has one or moreso-called risk factors, which are measurable parameters that correlatewith development of a condition described herein, which are describedherein. A subject having one or more of these risk factors has a higherprobability of developing a condition described herein than a subjectwithout these risk factor(s). In some embodiments, the subject is amammal. In some embodiments, the subject is a human. In someembodiments, the subject is a human diagnosed as having melanoma. Insome embodiments, the subject is a human suspected to have melanoma. Insome embodiments, the subject is a human having high risk of developingmelanoma. In some embodiments, the subject is a melanoma patient withmetastasis. In some embodiments, the subject is a melanoma patient withhigh risk of metastasis.

In some embodiments, methods of the present invention are used in thetreatment of melanoma. In some embodiments, the melanoma is one oflentigo maligna, lentigo maligna melanoma, superficial spreadingmelanoma, acral lentiginous melanoma, mucosal melanoma, nodularmelanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma,soft-tissue melanoma, melanoma with small nevus-like cells, melanomawith features of a Spitz nevus, uveal melanoma, or combinations thereof.

In some embodiments, the human patient has a melanoma at Stage 0, I, II,III or IV, or their respective subdivisions. In certain embodiments, themelanoma being treated is malignant metastatic melanoma. In certainembodiments, the melanoma being treated is stage I, stage II, stage IIIor stage IV. In other embodiments, the melanoma being treated is stageM1a, M1b or M1c melanoma. For detailed staging information, see Balch etal. (2001, “Final version of the American Joint Committee on Cancerstaging system for cutaneous melanoma”. J Clin Oncol 19 (16): 3635-48.PMID 11504745), which is incorporated by reference in its entirety.

In some embodiments, the human patient has one or more early signs ofmelanoma, such as changes to the shape or color of existing moles,including but not limited to, asymmetry, irregular borders, variegatedcolor, greater than about 6 mm in diameter, evolving over time, itch,ulcerate or bleed. In some embodiments, the human patient has nodularmelanoma, and early signs include, but are not limited to, theappearance of a new lump anywhere on the skin, elevated above the skinsurface, firm to the touch, and continuous growth.

Metastasis, or metastatic disease, is the spread of a cancer from oneorgan to another. Some cancer cells acquire the ability to penetrate thewalls of lymphatic and/or blood vessels, after which they are able tocirculate through the bloodstream to other sites and tissues in thebody. After the tumor cells come to rest at another site, theyre-penetrate the vessel or walls and continue to multiply, eventuallyforming another clinically detectable tumor.

In some embodiments, the melanoma is malignant metastatic melanoma. Insome embodiments, metastatic melanoma causes nonspecific paraneoplasticsymptoms in the patient, including but not limited to, loss of appetite,nausea, vomiting and fatigue. In some embodiments, the patient has brainmetastases. In some embodiments, the melanoma spread to the liver,bones, abdomen and/or distant lymph nodes.

In some embodiments, the subject to be treated has high risk ofdeveloping melanoma. In some embodiments, the human subject is aCaucasian. In some embodiments, the human patient is living in sunnyclimates with extensive exposure to UV light. In some embodiments, thesubject to be treated has one or more genetic mutations that increaseone's susceptibility to melanoma. In some embodiments, the geneticmutations are in the BRAF, MC1R, CDKN2A, CDK4, nucleotide excisionrepair (NER) enzymes (a.k.a. xeroderma pigmentosum, XP), multiple tumorsuppressor 1 (MTS1), and/or MDM2.

Methods for diagnosis of melanoma are well known, such as thosedescribed in Wurmand Soyer (October 2010, “Scanning for melanoma”.Australian Prescriber (33): 150-5), which is incorporated by referencein its entirety. In some embodiments, the diagnosis is by virtualexamination. In some embodiments, the diagnosis is by X-rays, CT scans,MRIs, PET and PET/CTs, ultrasound, LDH testing and/or photoacousticdetection.

After melanoma has been diagnosed, further tests can be used todetermine if cancer cells have spread within the skin or to other partsof the body. Tests include but are not limited to, physical exam andhistory, lymph node mapping and sentinel lymph node biopsy, CT scan, PETscan, MRI, and blood chemistry studies.

The terms “treating” and “treatment” as used herein refer to an approachfor obtaining beneficial or desired results including clinical results,and may include even minimal changes or improvements in one or moremeasurable markers of the disease or condition being treated. Atreatment is usually effective to reduce at least one symptom of acondition, disease, disorder, injury or damage. Exemplary markers ofclinical improvement will be apparent to persons skilled in the art.Examples include, but are not limited to, one or more of the following:decreasing the severity and/or frequency one or more symptoms resultingfrom the disease, diminishing the extent of the disease, stabilizing thedisease (e.g., preventing or delaying the worsening of the disease),delay or slowing the progression of the disease, ameliorating thedisease state, decreasing the dose of one or more other medicationsrequired to treat the disease, and/or increasing the quality of life,etc.

“Prophylaxis,” “prophylactic treatment,” or “preventive treatment”refers to preventing or reducing the occurrence or severity of one ormore symptoms and/or their underlying cause, for example, prevention ofa disease or condition in a subject susceptible to developing a diseaseor condition (e.g., at a higher risk, as a result of geneticpredisposition, environmental factors, predisposing diseases ordisorders, or the like).

In some embodiments, the methods comprise administering therapeuticallyeffective amount and/or prophylactically effective amount of acomposition comprising at least one immunostimulant to the subject. Theterm “therapeutically effective amount” as used herein, refers to thelevel or amount of one or more agents needed to treat a condition, orreduce or prevent injury or damage, optionally without causingsignificant negative or adverse side effects. A “prophylacticallyeffective amount” refers to an amount of an agent sufficient to preventor reduce severity of a future disease or condition when administered toa subject who is susceptible and/or who may develop a disease orcondition.

Immune stimulators, a.k.a. immunostimulants, or immunostimulators, referto substances that stimulate the immune system. In some embodiments, theimmune stimulators of the present invention can induce activation orincrease activity of one or more positive regulators of the immunesystem. In some embodiments, the immune stimulators of the presentinvention can induce deactivation or decrease activity of one or morenegative regulators of the immune system. As used herein, the termactivity refers to the activity of a given target at the genomic DNAlevel, transcriptional level, post-transcriptional level, translationallevel, post-translational level, including but not limited to, gene copynumber, mRNA transcription rate, mRNA abundance, mRNA stability, proteintranslation rate, protein stability, protein modification, proteinactivity, protein complex activity, etc. In some embodiments, an immunestimulator can be a specific immunostimulant. Specific immunositmulantsare substances that provide antigenic specificity in immune response,such as vaccines or antigens. In some embodiments, an immune stimulatorcan be a non-specific immunostimulant. Non-specific immunositmulants actirrespective of antigenic specificity to augment immune response ofother antigen or stimulate components of the immune system withoutantigenic specificity, such as adjuvants. An immune stimulator to beused in the methods of the present invention can be recombinant,synthetic, natural preparations, or combinations thereof.

In some embodiments, at least one immunostimulant is effective intreating sepsis.

In some embodiments, at least one immunostimulant is a thymosin peptide(thymosins). Thymosins are small proteins and are present in many animaltissues. Thymosins were originally isolated from the thymus, but mostare now known to be present in many other tissues. As used herein,thymosins include thymosin α, thymosin β, thymosin γ, and functionalvariants thereof. In certain embodiments, the thymosin is thymosin alpha(or alpha thymosin). In certain embodiments, the thymosin is prothymosinalpha (PTMA). In certain embodiments, the thymosin is thymosin alpha 1(“TA1”, a.k.a. Thymosin alpha-1, Thymosin a-1, Thymosin α-1, or alphathymosin) and functional variants having structural homology to TA1. Insome embodiments, TA1 is a peptide having the amino acid sequence(N-acetyl)-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(SEQ ID NO: 1). The amino acid sequence of TA1 is disclosed in U.S. Pat.No. 4,079,137, the disclosure of which is hereby incorporated byreference. TA1 is a non-glycosylated 28-amino acid peptide having anacetylated N-terminus, and a molecular weight of about 3108. In someembodiments, a synthetic version of TA1 is used. In some embodiments,the synthetic version of TA1 is commercially available in certaincountries under the trade name ZADAXIN (thymalfasin). As used herein,the term TA1 also refers to functional variants or fragments derivedfrom SEQ ID NO: 1.

In some embodiments, at least one immune stimulator is thymosin alpha 1(TA1). Alpha thymosin peptides comprise thymosin alpha 1 (TA1) peptidesincluding naturally occurring TA1 as well as synthetic TA1 orrecombinant TA1 having the amino acid sequence of naturally occurringTA1, amino acid sequences substantially similar thereto, or anabbreviated sequence form thereof, and their biologically active analogshaving substituted, deleted, elongated, replaced, or otherwise modifiedsequences which possess bioactivity substantially similar to that ofTA1, e.g., a TA1 derived peptide having sufficient amino acid homologywith TA1 such that it functions in substantially the same way withsubstantially the same activity as TA1. Suitable dosages of the alphathymosin peptide can be within the range of about 0.001-10 mg/kg/day. Insome embodiments, TA1 has the amino acid sequence disclosed in U.S. Pat.No. 4,079,137, the disclosure of which is incorporated herein byreference. TA1 initially isolated from Thymosin Fraction 5 (TF5) hasbeen sequenced and chemically synthesized. TA1 is a 28 amino acidpeptide with a molecular weight of 3108.

In some embodiments, effective amounts of an alpha thymosin peptide areamounts which may be dosage units within a range corresponding to about0.01-20 mg of TA1, about 1-10 mg of TA1, about 2-10 mg of TA1, about 2-7mg of TA1, or about 3-6.5 mg of TA1, and may comprise about 1.6, 3.2 or6.4 mg of TA1, or about 3.2 or 6.4 mg of TA1. A dosage unit may beadministered once per day, or a plurality of times per day. In someembodiments, TA1 is administered to a subject at a dosage within a rangeof about 0.5-10 mg/day. In certain embodiments, the TA1 dosage is withina range of about 1.5-7 mg/day, or within a range of about 1.6-6.4mg/day. In certain embodiments, the TA1 dosage is within a range ofabout 1.7-10 mg/day, about 1.7-7 mg/day, or about 3-7 mg/day. In someembodiments, the effective dosages include about 1.6, 3.2 or 6.4 mg/day.In some embodiments, TA1 is administered to a subject at a dosage ofabout 0.01 to about 6 mg/kg. In some embodiments, TA1 is administered toa subject once a day, twice a day, three times a day, four times a day,or more. In some embodiments, TA1 is administered to a subject alone orwith one or more additional immune stimulators.

TA1 peptides include naturally occurring TA1 as well as synthetic TA1 orrecombinant TA1 having the amino acid sequence of naturally occurringTA1, amino acid sequences substantially similar thereto, or anabbreviated sequence form thereof, and their biologically active analogshaving substituted, deleted, elongated, replaced, or otherwise modifiedsequences which possess bioactivity substantially similar to that ofTA1, e.g., a TA1 derived peptide having sufficient amino acid homologywith TA1 such that it functions in substantially the same way withsubstantially the same activity as TA1. In some embodiments, suitabledosages of the thymosin can be within the range of about 0.001-10mg/kg/day, such as 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06,0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0,3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, or more mg/kg/day. In someembodiments, TA1 has the amino acid sequence disclosed in U.S. Pat. No.4,079,137, the disclosure of which is incorporated herein by reference.

TA1 initially isolated from Thymosin Fraction 5 (TF5) has been sequencedand chemically synthesized. TA1 is a 28 amino acid peptide with amolecular weight of 3108. The term TA1 also includes functional variantsand functional fragment of TA1, naturally occurring, synthetic orrecombinant thymosin. TA1 was originally isolated from bovine thymus,where it was shown to reconstitute “immune function” in thymectomizedanimal models. In some embodiments, the thymosin comprises the aminoacid sequence of SEQ ID NO:1 (where an acylated, e.g., acetylated,N-terminus is optional). In some embodiments, the thymosin comprises anamino acid sequence that is substantially similar to TA1, and maintainsthe immunomodulatory activity of TA1. The substantially similar sequencemay have, for example, from about 1 to about 10 amino acid deletions,insertions, and/or substitutions (collectively) with respect to TA1. Forexample, the thymosin may have from about 1 to about 5 (e.g., 1, 2, or3) amino acid insertions, deletions, and/or substitutions (collectively)with respect to TA1.

Thus, the thymosin may comprise an abbreviated TA1 sequence, forexample, having deletions of from 1 to about 10 amino acids, or fromabout 1 to 5 amino acids, or 1, 2 or 3 amino acids with respect to TA1.Such deletions may be at the N- or C-terminus, and/or internal, so longas the immunomodulatory activity of the peptide is substantiallymaintained. Alternatively, or in addition, the substantially similarsequence may have from about 1 to about 5 amino acid insertions (e.g.,1, 2, or 3 amino acid insertions) with respect to TA1, where theimmunomodulatory activity of TA1 is substantially maintained.Alternatively, or in addition, the substantially similar sequence mayhave from 1 to about 10 amino acid substitutions, where theimmunomodulatory activity is substantially maintained. For example, thesubstantially similar sequence may have from 1 to about 5, or 1, 2, or 3amino acid substitutions, which may include conservative andnon-conservative substitutions. In some embodiments, the substitutionsare conservative. Generally, conservative substitutions includesubstitutions of a chemically similar amino acid (e.g., polar,non-polar, or charged). Substituted amino acids may be selected from thestandard 20 amino acids or may be a non-standard amino acid (e.g., aconserved non-standard amino acid).

In some embodiments, the thymosin comprises an amino acid sequencehaving at least 70% sequence identity to SEQ ID NO:1, while maintainingthe immunomodulatory activity of TA1. For example, the thymosin maycomprise an amino acid sequence having at least 80%, 85%, 90%, 95%sequence identity to SEQ ID NO:1. The thymosin may comprise an aminoacid sequence having 100% sequence identity to SEQ ID NO:1. In someembodiments, the N-terminus may be optionally acylated (e.g.,acetylated) or alkylated, for example, with a C1-10 or C1-C7 acyl oralkyl group.

In certain embodiments, the substantially similar and homologouspeptides described above may function at a level of at least about 50%,70%, 80%, 90%, or about 100% relative to TA1 (SEQ ID NO:1).

The thymosin may be prepared synthetically, for example, by solid phasesynthesis, or may be made recombinantly and purified by knowntechniques. The thymosin may also be provided in lyophilized form, andreconstituted with sterile (e.g., aqueous) diluent prior toadministration. Formulations of thymosin may be administered bysubcutaneous injection, or other effective route.

In certain embodiments, the thymosin is pegylated to increase itshalf-life in circulation. Such strategies for increasing the half-lifeof therapeutic proteins are well known.

Thymosin is thought to play a role in inflammatory and innate immuneresponses, and to facilitate discrimination of self from non-self inmammals. Activation of PAMP (pathogen-associated molecular patterns)ligands by thymosin leads to stimulation of intracellular signaltransduction pathways resulting in expression of co-stimulatorymolecules, pro-inflammatory cytokines, nitric oxide, and eicosanoids.Thymosin may affect, for example, dendritic cells, T cells, B cells, andNK cells.

In some embodiments, TA1 is combined with a second immune stimulator.

In some embodiments, the second immune stimulator is an immunestimulator that is effective in treating sepsis. In some embodiments,the second immune stimulator is GM-CSF, interferon (e.g., interferon-γ),interleukin 7, interleukin 15, or an inhibitor of PD-1. In someembodiments, the immune stimulator that is effective in treating sepsisis capable of reducing T-cell exhaustion in the subject. In someembodiments, the immune stimulator is a substance capable of increasingthe activity of GM-CSF, interferon (e.g., interferon-γ), or interleukin7 or interleukin 15, see Boomer (“The changing immune system in sepsis:Is individualized immuno-modulatory therapy the answer?”, Virulence 5:1,45-56; Jan. 1, 2014), which is incorporated by reference in itsentirety.

In some embodiments, the second immune stimulator is an inhibitor to acheckpoint protein (a.k.a. checkpoint inhibitor, immune checkpointmodulators, or CPMs). As used herein, a checkpoint protein is one thatkeeps the immune system from attacking the cells. Checkpoint inhibitorsare designed to lessen the effectiveness of checkpoint proteins. In someembodiments, the checkpoint proteins include, but are not limited to,PD1, PDL1, CTLA4, KIR, IDO1, 4-1BB (CD137), OX40 (CD134), and LAG3.

In some embodiments, the second immunostimulants are capable ofattenuating abnormal immune suppression in the subject. In someembodiments, the abnormal immune suppression is due to abnormally highactivity of an immune suppressor in the immune system. In someembodiments, the immune suppressor with abnormally high activity in thesubject is programmed death receptor (PD-1), programmed death ligand(PD-L), B and T lymphocyte attenuator (BTLA), herpesvirus entry mediator(HVEM), or cytokine IL-10. In some embodiments, the secondimmunestimulator effective in treating sepsis is an inhibitor of theimmune suppressor that has abnormally high activity in a sepsis patientduring the hypo-inflammatory phase, see Boomer (“The changing immunesystem in sepsis: Is individualized immuno-modulatory therapy theanswer?”, Virulence 5:1, 45-56; Jan. 1, 2014), which is hereinincorporated by reference in its entirety for all purposes. In someembodiments, the second immune stimulator is an inhibitor of PD-1, PD-L,BTLA, HVEM and/or IL-10. In some embodiments, the inhibitor reduces theactivity of PD-1, PD-L, BTLA, HVEM and/or IL-10 at DNA level, mRNAlevel, and/or protein level. In some embodiments, the inhibitor is anantibody against PD-1, PD-L, BTLA, HVEM or IL-10. In some embodiments,the inhibitor is an antibody against PD-1, such as those described inU.S. Pat. Nos. 8,552,154, 8,741,295, 8,008,449, 8,460,886 and 7,029,674,or U.S. Patent Application Publication Nos. 20110171220, 20110271358,20140044738, each of which is herein incorporated by reference in itsentirety. In some embodiments, the inhibitor is an antibody against thePD ligand. In some embodiments, the inhibitor inhibits the interactionbetween PD-1 and its ligand.

In some embodiment, the PD-1 inhibitor is an antibody against PD-1. Insome embodiments, the dosage of the PD-1 antibody is about 0.1 to 10mg/kg, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10 mg/kg. In someembodiments, the dosage of PD-1 antibody is about 1-5 mg/kg, or about2-3 mg/kg.

As used herein, the phrase “an inhibitor of PD-1” refers to a compoundthat inhibits the signaling pathway mediated by PD-1, such as aninhibitor to a component in the PD-1 signaling pathway. The PD-1signaling pathway is described in Riley (Immunol Rev. 2009 May; 229(1):114-125.), which is herein incorporated by reference in its entirety. Asused herein, the term “activity” of a component in the PD-1 signalingpathway can be a parameter at genomic DNA level, transcriptional level,post-transcriptional level, translational level, post-translationallevel, including, but not limited to gene activity, RNA activity, andprotein activity. The gene activity can be gene copy number, geneamplification number, or promoter activity, etc. RNA activity can bemRNA abundance, synthesis rate, and/or stability, etc. Protein activitycan be protein abundance, synthesis rate, stability, enzymatic activity,phosphorylation rate, modifications, binding activity, etc. In someembodiments, the inhibitors reduce the activity of PD-1. In someembodiments, the inhibitors reduce the activity of a ligand for PD-1. Insome embodiments, the inhibitor is a PD-1 inhibitor, such as ananti-PD-1 antibody, or an inhibitor of the ligand for PD-1 (a.k.a.PDL-1), such as an anti-PDL-1 antibody. The antibody can be eithermonoclonal, polyclonal, or a combination thereof.

In some embodiments, the second immune stimulator is a cytokine. In someembodiments, cytokines include, but are not limited to, chemokines,interferons, interleukins, lymphokines, tumour necrosis factor.

In some embodiments, the cytokine as the second immune stimulator is acolony-stimulating factor (CSF). As used herein, the term CSF refers toisolated, synthetic, or recombinant CSFs, including functionalderivatives and functional fragments thereof. As used herein, the termCSF refers to substances comprising either a full length colonystimulating factor polypeptide, functional fragment thereof, and/orfunctional derivatives thereof. Colony stimulating factors are secretedglycoproteins that bind to receptor proteins on the surfaces ofhemopoietic stem cells, thereby activating intracellular signalingpathways that lead to cell proliferation and/or differentiation intospecific kind of blood cells, such as white blood cells.

In some embodiments, the CSF comprise a polypeptide of macrophagecolony-stimulating factors (e.g., CSF1, or M-CSF), granulocytemacrophage colony-stimulating factors (e.g., CSF2, a.k.a. GM-CSF),granulocyte colony-stimulating factors (e.g., CSF3, a.k.a. GCSF, orG-CSF), and/or analogs thereof, such as promegapoietin or filgrastim, ora functional fragment thereof capable of stimulate immune system in asubject.

In some embodiments, the cytokine as the second immune stimulator isGM-CSF. As used herein, the term GM-CSF refers to isolated, synthetic,or recombinant GM-CSFs, including functional derivatives and functionalfragments thereof. Naturally, GM-CSF can be secreted by macrophages, Tcells, mast cells, NK cells, endothelial cells and fibroblasts. In someembodiments, the immune stimulator can be pharmaceutical analogs ofnatural GM-CSF, such as sargramostim and molgramostim. In someembodiments, the GM-CSF is in the form of homodimer or heterodimer. Insome embodiments, the GM-CSF is manufactured using recombinanttechnology (e.g., molgramostim or sargramostim (a.k.a., leukine)).

In some embodiment, the dosage of GM-CSF is about 1 to 1000 mcg/m², suchas about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000 mcg/m². In some embodiments, the dosage ofGM-CSF is about 125 to about 250 mcg/m².

In some embodiments, the cytokine as the second immune stimulator is aninterferon. As used herein, the term interferon refers to isolated,synthetic, or recombinant interferons, including functional derivativesand functional fragments thereof. As used herein, interferons (IFNs)refer to polypeptides made and released by host cells in response to thepresence of pathogens, such as viruses, bacteria, parasites or tumorcells. In some embodiments, the interferon activates immune cells. Insome embodiments, the interferon activates natural killer cells andmacrophages. In some embodiments, the interferon increases the activityof major histocompatibility complex (MHC) antigents. In someembodiments, the interferon belongs to Type I IFN, Type II IFN, or TypeIII IFN. In some embodiments, the Type I IFN is IFN-α, IFN-β, IFN-ε,IFN-κ, or IFN-{acute over (ω)}. In some embodiments, the Type II IFNbinds to IFNR that consists of IFNFGR1 and IFNGR2 chains, such as IFN-γ.In some embodiments, the Type III IFN signals through a receptor complexconsisting of CRF2-4 and IFNLLR1. In some embodiments, an interferon ofthe present application increase the activity of MHC I and/or MHC IIactivity. In some embodiments, the interferon increases theimmunoproteasome activity in the subject. In some embodiments, theinterferon increases the activity of cytotoxic T cells. In someembodiments, the interferon activates signal transducer and activator oftranscription (STAT) complexes. In some embodiments, the interferonactivates Janus kinase-STAT (JAK-STAT) signaling pathway. In someembodiments, the interferon activates the CRK family of adaptor proteinCRKL, which is a nuclear adaptor for STATS that also regulates signalingthrough the C3G/Rap1 pathway. In some embodiments, the interferonactivates the p38 mitogen-activated protein kinase (MAP kinase) toinduce gene transcription. In some embodiments, the interferon activatesthe phosphatidylinositol 3-kinase (PI3K) signaling pathway. In someembodiments, the interferon increases the activity of helper T cells. Insome embodiments, the interferon is IFN-γ. In some embodiments, theinterferons directly activate macrophages and/or natural killer cells.In some embodiments, the immune stimulator can induce interferons. Insome embodiments, the interferon is linked to polyethylene glycol.

In some embodiments, the cytokine as the second immune stimulator is atumor necrosis factor (TNF). As used herein, the term TNF refers toisolated, synthetic, or recombinant TNF, including functionalderivatives and functional fragments thereof. In some embodiments, theTNF can be produced by activated macrophages, CD4+ lymphocytes, NKcells, neutrophils, mast cells, eosinophils, or neurons.

In some embodiments, the cytokine as the second immune stimulator is aninterleukin. As used herein, the term interleukin refers to isolated,synthetic, or recombinant interleukins, including functional derivativesand functional fragments thereof. In some embodiments, the interleukincan be synthesized by helper CD4 T lymphocytes, monocytes, macrophages,or endothelial cells. In some embodiments, the interleukin promotes thedevelopment and/or differentiation of T lymphocytes, B lymphocytes,and/or hematopoietic cells. In some embodiments, the immune stimulatorcomprises interleukin 1, interleukin 3, interleukin 4, interleukin 5,interleukin 6, interleukin 7, interleukin 8, interleukin 9, interleukin10, interleukin 11, interleukin 12, interleukin 13, interleukin 14,interleukin 15, interleukin 16, or interleukin 17. As used herein, theterm interleukin refers to both interleukin isolated, synthetic, orrecombinant interleukins, including functional derivatives andfunctional fragments thereof. In some embodiments, the immune stimulatorcomprises IL-7, IL-9 and/or IL-15. In some embodiments, the immunestimulator comprises an interleukin that can serve as a growth factorfor lymphoid cells, such as B-cell lineages, T-cell lineages and/or NKcells. In some embodiments, the immune stimulator comprises aninterleukin that can support growth of helper T cells. In someembodiments, the immune stimulator comprises an interleukin that canstimulate and maintain cellular immune responses. In some embodiments,the immune stimulator comprises an interleukin that can stimulate theproliferation of lymphoid cells, such as B-cell lineages and/or T-celllineages.

In some embodiments, the second immune stimulator comprises a substancethat can enhance the activity of an IL receptor. In some embodiments,the IL receptor is the IL-7 receptor. In some embodiments, the immunestimulator comprises a substance that can enhance the interaction ofIL-7 and IL-7 receptor. In some embodiments, the immune stimulatorcomprises Interleukin-7 receptor alpha. In some embodiments, the immunestimulator comprises common gamma chain receptor, which formsheterodimer with Interleukin-7 receptor alpha. In some embodiments, theIL receptor is the IL-9 receptor. In some embodiments, the immunestimulator comprises a substance that can enhance the interaction ofIL-9 and IL-9 receptor. In some embodiments, the immune stimulatorcomprises Interleukin-9 receptor. In some embodiments, the IL receptoris the IL-15 receptor. In some embodiments, the immune stimulatorcomprises a substance that can enhance the interaction of IL-15 andIL-15 receptor. In some embodiments, the immune stimulator comprisesInterleukin-15 receptor beta chain (CD122). In some embodiments, theimmune stimulator comprises Interleukin-15 receptor common gamma chain(gamma-C, CD132).

In some embodiments, the second immune stimulator comprises aninterleukin. In some embodiments, the interleukin is IL-7. In someembodiment, the dosage of IL-7 is about 0.1 to 100 mcg/kg, such as about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mcg/kg. In some embodiment,the dosage is about 1 to 50 mcg/kg, or about 3 to 30 mcg/kg.

Other immune stimulators can be used. In some embodiments, the immunestimulator can function as a white blood cell growth factor. In someembodiments, the immune stimulator stimulates stem cells to producegranulocytes (e.g., neutrophils, eosinophils, and basophils) and/ormonocytes. In some embodiments, the immune stimulator can preventneutropenia following chemotherapy. In some embodiments, the immunestimulator can stimulates the survival, proliferation, differentiation,and/or function of neutrophil precursors and mature neutrophils. In someembodiments, the immune stimulator functions by using one or moresignaling pathways including but not limited to, Janus kinase (JAK),signal transducer and activator of transcription (STAT),Ras/mitogen-activated protein kinase (MAPK), phosphatidylinositol3-kinase (PI3K), and protein kinase B (Akt) signal transduction pathway.

In some embodiments, an immunostimulant or a combination of at least twoimmunostimulants of the present application is combined with ananti-cancer agent.

In some embodiments, an anti-cancer agent that may be used incombination with a immunostimulant of the present application mayinclude, but are not limited to, estrogen receptor antagonist, receptortyrosine kinase inhibitors, cancer cell replication inhibitors, cancercell signaling inhibitors or silences and other inhibitors of tumor cellsurface of internal cell signaling molecules implicated in cancer cellgrowth, cancer cell resistance to apoptosis and/or cancer cellmetastases.

In some embodiments, a immunostimulant of the present application isused in combination with an estrogen receptor antagonist (ERANT), aninhibitor to the estrogen receptor, or an inhibitor to the estrogenreceptor ligand.

In some embodiments, a immunostimulant of the present application isused in combination with a receptor tyrosine kinase inhibitor. Tyrosinekinase inhibitors represent a class of therapeutic agents or drugs thattarget receptor and/or non-receptor tyrosine kinases in cells such astumor cells. In certain instances, the tyrosine kinase inhibitor is anantibody-based (e.g., anti-tyrosine kinase monoclonal antibody, etc.) orpolynucleotide-based (e.g., tyrosine kinase antisense oligonucleotide,small interfering ribonucleic acid, etc.) form of targeted therapy. Insome embodiments, the tyrosine kinase inhibitor is a small molecule thatinhibits target tyrosine kinases by binding to the ATP-binding site ofthe enzyme.

In some embodiments, a immunostimulant of the present application isused in combination with a cancer cell replication inhibitor, such asanti-microtubule agents, which refer to chemicals that block celldivision by preventing microtubule function.

In some embodiments, a immunostimulant of the present application isused in combination with a cancer cell signaling inhibitor. In someembodiments, the cancer cell signaling inhibitor include agents that caninhibit EGFR (epidermal growth factor receptor) responses, such as EGFRantibodies, EGF antibodies, and molecules that are EGFR inhibitors; VEGF(vascular endothelial growth factor) inhibitors; and erbB2 receptorinhibitors, such as organic molecules or antibodies that bind to theerbB2 receptor.

In some embodiments, the additional anti-cancer agent can beadministered prior to, concurrently, or after the administration of afirst immune stimulator of the present invention.

In some embodiments, the anti-cancer agent comprises ipilimymab orderivatives as described in U.S. Pat. Nos. 7,611,702, 7,741,345, and8,088,770, which are incorporated by reference in their entireties.

In some embodiments, the anti-cancer agent comprises a signaltransduction inhibitor. In some embodiments, the transduction inhibitoris a BRAF inhibitor, such as vemurafenib and dabrafenib. In someembodiments, the transduction inhibitor is a MEK inhibitor, such astrametinib. In some embodiments, the transduction inhibitor is a c-KITinhibitor, such as imatinib.

In some embodiments, the anti-cancer agent comprises a kinase inhibitor.In some embodiments, the kinase inhibitor comprises sorafenib orderivatives as described in U.S. Pat. No. 7,235,576. The kinaseinhibitor may be administered continuously (i.e., daily), multiple timesper day, every other day, etc., and may be administered prior to,concurrently, or after administration of an immune stimulator of thepresent, e.g., on the same day(s) or on different days during the courseof the treatment regimen. In certain embodiments, the kinase inhibitoris administered in a dosage range of, e.g., about 10-2000 mg/day ofadministration, about 50-1000 mg/day, or about 50-800 mg/day. Dailydosages may be, e.g., about 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500mg, 600 mg, 700 mg, 800 mg, etc.

In some embodiments, the anti-cancer agent comprises an antineoplasticheat shock apoptosis activator (HSAA), see US Patent ApplicationPublication No. 20100317583, which is herein incorporated by referencein its entirety. In some embodiments, the HSAA comprises STA-4783(elesclomol). The HSAA may be administered continuously (i.e., daily),multiple times per day, every other day, etc., and may be administeredprior to, concurrently, or after administration of an immune stimulatorof the present, e.g., on the same day(s) or on different days during thecourse of the treatment regimen. In certain embodiments, the HSAA isadministered in dosage ranges of, e.g., about 0.01-1000 mg/kg/day ofadministration, about 0.1-500 mg/kg/day, or about 1-200 mg/kg/day. Dailydosages may be, e.g., 25 mg/kg, 100 mg/kg, etc.

In some embodiments, the anti-cancer agent comprises an inhibitoragainst cytotoxic T lymphocyte-associated antigen 4 (CTLA4), see USPatent Application Publication No. 20100330093, which is hereinincorporated by reference in its entirety. In some embodiments, theinhibitor is an antibody against CTLA4. In some embodiments, the CTLA4antibodies include, but are not limited to, 9H10 (EBIOSCIENCE), MDX010(MEDAREX), 1F4 (GENETEX), BNI3 (GENETEX), Q01 (ABNOVA), A01 (ABNOVA),M08 (ABNOVA), 1B8 (ABCAM), WKH203 (ABCAM), ab9984 (ABCAM), ab13486(ABCAM), ipilimumab, ticilimumab or a combination thereof. In someembodiments, the CTLA4 antibodies may be administered continuously(i.e., daily), multiple times per day, every other day, etc., and may beadministered prior to, concurrently, or after administration of animmune stimulator of the present, e.g., on the same day(s) or ondifferent days during the course of the treatment regimen. In someembodiments, the CTLA4 antibodies are administered in a dosage range of,e.g., 0.001-50 mg/kg patient body weight per day of administration, orabout 0.01-20 mg/k, or about 1-15 mg/kg.

In certain embodiments, the anti-cancer agent comprises one or moreantineoplastic agents. In some embodiments, the antineoplastic agentsare chemotherapeutics. In some embodiments, the chemotherapeutics areselected from alkylating agents, anti-metabolites, anti-microtubuleagents, Topoisomerase inhibitors, and Cytotoxic antibiotics.

As used herein, the term “alkylating agents” refers to agents that havethe ability to alhylate molecules in a subject, including proteins, RNAand DNA. Non-limiting examples of alkylating agents include nitrogenmustards, nitrosoureas, tetrazines, aziridines, cisplatins andderivatives, and non-classical alkylating agents. Nitrogen mustardsinclude mechlorethamine, cyclophosphamide, melphalan, chlorambucil,ifosfamide and busulfan.

As used herein, the term “anti-metabolites” refers to molecule thatimpedes DNA, RNA, or protein synthesis. In some embodiments,anti-metabolites resemble either nucleobases or nucleosides (anucleotide without the phosphate group), but have altered chemicalgroups. These drugs exert their effect by either blocking the enzymesrequired for DNA synthesis or becoming incorporated into DNA or RNA. Byinhibiting the enzymes involved in DNA synthesis, they prevent mitosisbecause the DNA cannot duplicate itself. Also, after misincorperation ofthe molecules into DNA, DNA damage can occur and programmed cell death(apoptosis) is induced. In some embodiments, the anti-metabolites areanti-folates, fluoropyrimidines, deoxynucleoside analogues andthiopurines. In some embodiments, the anti-metabolites are selected frommethotrexate, pemetrexed, fluorouracil, capecitabine, cytarabine,gemcitabine, decitabine, Vidaza, fludarabine, nelarabine, cladribine,clofarabine, pentostatin, thioguanine and mercaptopurine.

As used herein, the term “anti-microtubule agents” refers to chemicalsthat block cell division by preventing microtubule function.

In some embodiments, the anti-tumor agents are mitotic inhibitors.

As used herein, the term “topoisomerase inhibitors” refers to agentsthat can modulate the activity of topoisomerase I and/or topoisomeraseII. In some embodiments, the topoisomerase inhibitor of this inventioncan be a topoisomerase I inhibitor. In further embodiments of thisinvention, the topoisomerase inhibitor is a topoisomerase II inhibitor.

As used herein, the term “cytotoxic antibiotics” cytotoxic antibioticsinclude, but are not limited to, antinomycin, bleomycin, mitomycin,plicamycin and the like. Examples of tyrosine kinase inhibitors include,but are not limited to, nilotinib, imatinib, gefitinib, erlotinib,cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzuman and thelike.

In some embodiments, the other anti-cancer agents are monoclonalantibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab,rituximab, and trastuzumab; photosensitizers, such as aminolevulinicacid, methyl aminolevulinate, porfimer sodium, and verteporfin; andother agents, such as alitretinoin, altretamine, amsacrine, anagrelide,arsenic trioxide, asparaginase, bexarotene, bortezomib, celecoxib,denileukin diftitox, erlotinib, estramustine, gefitinib,hydroxycarbamide, imatinib, pentostatin, masoprocol, mitotane,pegaspargase, and tretinoin.

In some embodiments, the antineoplastic agent comprises alkylatingantineoplastic agents (AIkAA). In some embodiments, the AIkAA comprisesdacarbazine (DTIC). In some embodiments, an alkylating antineoplasticagent may be administered to patient within a dosage range of, e.g.,about 700-1300 mg/m2/day, more preferably in a dosage range of about800-1200 mg/m2/day, and most preferably about 1000 mg/m2/day.

In some embodiments, a pharmaceutical composition of the presentinvention can ameliorate, treat, and/or prevent one or more symptoms ofmelanoma in a clinically relevant, statistically significant and/orpersistent fashion. In some embodiments, administration of apharmaceutical composition of the present invention providesstatistically significant therapeutic effect for ameliorating, treating,and/or preventing one or more symptoms of melanoma. In one embodiment,the statistically significant therapeutic effect is determined based onone or more standards or criteria provided by one or more regulatoryagencies in the United States, e.g., FDA or other countries. In someembodiments, the statistically significant therapeutic effect isdetermined based on results obtained from regulatory agency approvedclinical trial set up and/or procedure. In some embodiments, apharmaceutical composition of the present invention providesstatistically significant therapeutic effect as measured byrecurrence-free survival (RFS, the length of time before recurrence ordeath). In some embodiments, a pharmaceutical composition of the presentinvention provides statistically significant therapeutic effect asmeasured by frequency and/or severity of metastases.

In some embodiments, the statistically significant therapeutic effect isdetermined based on a patient population of at least 50, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, or more. In some embodiments, thestatistically significant therapeutic effect is determined based on dataobtained from randomized and double blinded clinical trial set up. Insome embodiments, the statistically significant therapeutic effect isdetermined based on data with a p value of less than or equal to about0.05, 0.04, 0.03, 0.02 or 0.01. In some embodiments, the statisticallysignificant therapeutic effect is determined based on data with aconfidence interval greater than or equal to 95%, 96%, 97%, 98% or 99%.In some embodiments, the statistically significant therapeutic effect isdetermined on approval of Phase III clinical trial of the methodsprovided by the present invention, e.g., by FDA in the US.

In some embodiment, the statistically significant therapeutic effect isdetermined by a randomized double blind clinical trial of a patientpopulation of at least 50, 100, 200, 300 or 350; treated with apharmaceutical composition of the present invention, but not incombination with any other agent for treating MD symptoms. In someembodiment, the statistically significant therapeutic effect isdetermined by a randomized clinical trial of a patient population of atleast 50, 100, 200, 300 or 350 and using any commonly accepted criteriafor MD symptoms assessment, such as the criteria described herein.

In general, statistical analysis can include any suitable methodpermitted by a regulatory agency, e.g., FDA in the US or China or anyother country. In some embodiments, statistical analysis includesnon-stratified analysis, log-rank analysis, e.g., from Kaplan-Meier,Jacobson-Truax, Gulliken-Lord-Novick, Edwards-Nunnally, Hageman-Arrindeland Hierarchical Linear Modeling (HLM) and Cox regression analysis.

In some embodiments, the methods comprise administering the immunestimulator at a specific phase of the melanoma progression. In someembodiments, the immune stimulator is administered to the subject whenapoptosis of T-cells in the subject starts. Methods of detectingapoptosis of T-cells are well known, such as those using FITC Annexin V.In some embodiments, the immune stimulator is administered to thesubject when the subject experiences T-cell exhaustion due to apoptosisof T-cells. Methods of T-cells quantification are well known, such asthose using flow cytometry. In some embodiments, the immune stimulatoris administered to the subject in order to maintain a predeterminedlevel of active T-cell populations in the subject. In some embodiments,the activated T-cells are CD8+ T-cells and/or CD4+ T-cells.

In certain embodiments, the treatment regimen comprises a plurality ofdays of a pharmaceutical composition comprising an immune stimulator ofthe present invention, and the immune stimulator can be administered tothe subject during at least a portion of the treatment regimen.

In certain embodiments, the treatment regimen comprises administeringthe pharmaceutical composition for a period of about 1-10 days, about1-20 days, about 1-30 days, about 1-40 days, about 1-50 days, about 1-60days, about 1-70 days, about 1-80 days, about 1-90 days, about 1-100days, or more.

In certain embodiments, the treatment regimen further comprises about1-5 days, about 5-10 days, about 10-20 days, about 20-30 days or more ofnon-administration of the pharmaceutical composition. In someembodiments, the pharmaceutical composition may be administered dailyfor 1-10 days, about 1-20 days, about 1-30 days, about 1-40 days, about1-50 days, about 1-60 days, about 1-70 days, about 1-80 days, about 1-90days, about 1-100 days, or more, followed by about 1-5 days, about 5-10days, about 10-20 days, about 20-30 days of non-administration of thealpha thymosin peptide.

In some embodiments, the methods further comprise monitoring theresponse of the subject after administration to avoid severe and/orfatal immune-mediated adverse reactions due to over-activation andproliferation. In some embodiments, the administration of the immunestimulator is modified, such as reduced, paused or terminated if thepatient shows persistent moderate adverse reactions. In someembodiments, the dosage is modified if the patient fails to respondwithin about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14weeks, 15 weeks, 16 weeks or more from administration of first dose. Insome embodiments, the dosage is modified if the patient shows severe orlife-threatening adverse reactions, including but not limited to,colitis with abdominal pain, fever, ileus, or peritoneal signs; increasein stool frequency (≧7 over baseline), stool incontinence, need forintravenous hydration for >24 hours, gastrointestinal hemorrhage, andgastrointestinal perforation, AST or ALT>5× the upper limit of normal(ULN) or total bilirubin>3× the ULN, Stevens-Johnson syndrome, toxicepidermal necrolysis, or rash complicated by full-thickness dermalulceration or necrotic, bullous, or hemorrhagic manifestations, severemotor or sensory neuropathy, Guillain-Barré syndrome, myasthenia gravis,severe immune-mediated reactions involving any organ systemimmune-mediated ocular disease which is unresponsive to topicalimmunosuppressive therapy.

In some embodiments, the methods comprise determining the activity ofone or more components in the immune system before, during, and/or afteradministration of an immune stimulator of the present invention. In someembodiments, a treatment regimen of the present invention can bemodified based on the activity of one or more components in the immunesystem. In some embodiments, the components in the immune systemincludes, but are not limited to T-cell apoptosis, CD+8 T-cells, andCD+4 T-cells. In some embodiments, the methods comprise determining oneor more biomarkers indicating the activity of T-cell apoptosis, CD+8T-cells, and/or CD+4 T-cells. In some embodiments, the methods comprisedetermining the activity of effector T cells. In some embodiments, themethods comprise determining the activity of helper T cells.

In some embodiments, the activity of one or more components in theimmune system subject is compared to a pre-determined standard to decideif a pharmaceutical composition of the present invention should beadministered to the subject and/or when the pharmaceutical compositioncan be administered to the subject. In some embodiments, the componentcan be IL-2, IL-2 receptor, IL-7, IL-7 receptor, IL-15, IL-15 receptor,CD69, IFNγ, IL-6, TNF, IL-1, GM-CSF, PD-L, PD-1, IL-10, BTLA, HVEM,IL-1β, IL-4, IL-6, IL-10, or combinations thereof. In some embodiments,a pharmaceutical composition of the present invention is administered toa subject when the activity of PD-L, PD-1, IL-10 TLA, and/or HVEM ishigher compared to the pre-determined standard. In some embodiments, apharmaceutical composition of the present invention is administered to asubject when the activity of IL-2, IL-2 receptor, IL-7, IL-7 receptor,IL-15, IL-15 receptor, CD69, IFNγ, IL-6, TNF, and/or GM-CSF is lowercompared to the pre-determined standard. In some embodiments, apharmaceutical composition of the present invention is administered to asubject when the activity of IL-1β is higher compared to thepre-determined standard. In some embodiments, a pharmaceuticalcomposition of the present invention is administered to a subject whenthe activity of IL-4 is lower compared to the pre-determined standard.In some embodiments, a pharmaceutical composition of the presentinvention is administered to a subject when the activity of IL-6 ishigher compared to the pre-determined standard. In some embodiments, apharmaceutical composition of the present invention is administered to asubject when the activity of IL-10 is higher compared to thepre-determined standard.

In some embodiments, treatment methods of the present invention arecombined with one or more additional treatments for cancer. In someembodiments, the additional treatment is surgery. In some embodiments,the additional treatment is an adjuvant treatment. In some embodiments,the additional treatment is chemotherapy. In some embodiments, theadditional treatment is immunotherapy. In some embodiments, theadditional treatment is radiation therapy. In some embodiments, theadditional treatment is a targeted therapy, such as adoptive celltherapy or gene therapy.

In certain embodiments, the subject is immunodeficient. Animmunodeficient subject (e.g., a human subject) exhibits a reducedcapacity to fight infectious disease and/or a reduced capacity torespond to pathogen exposure. Examples of such immunodeficient subjectsinclude an elderly patient, newborn, leukemic or neutropenic patient, apatient on hemodialysis (e.g., for treatment of chronic renal disease),patient receiving immunosuppressant therapy, AIDS patient, diabeticpatient, patient receiving chemotherapy or radiation therapy for cancer,immunodeficiency caused by a genetic defect, malnutrition, drug abuse,alcoholism, or other immune-compromising illness or condition.

In certain embodiments, the immune-compromised subject is elderly. Asanimals age, their immune response is reduced, and the robustness of theimmune response is diminished due to the prevalence of low affinityantibody response. Accordingly, the subject in these embodiments may bea human patient over the age of 45, or over the age of 50. In someembodiments, the subject is a human patient 60 years of age or older, 65years of age or older, or 70 years of age or older.

In certain embodiments, the treatment regimen further comprisesdetermine the patient response during the treatment. In someembodiments, one or more symptoms associated with the infection areevaluated to determine the subject's response to the treatment regimen.

In some embodiments, a composition of the present invention induces astrong and rapid immune response to pathogens in the subject or thepopulation of subjects. The regimen of thymosin as described hereinprovides the patient with a more robust immune response to pathogenexposure, including but not limited to, higher antibody titers and/or amore rapid antibody response. In some embodiments, the regimen providessuch advantages for up to about 10 days, 20 days, 30 days, 40 days, 50days or more with as few as one, two, three, or four administrations.

In some embodiments, the subject has been diagnosed as having a cancer.In some embodiments, the cancer is melanoma.

In some embodiments, a composition of the present invention isadministered by any suitable methods known in the art. In someembodiments, administration of a composition of the present inventionmay be carried out orally, parenterally, subcutaneously, intravenously,intramuscularly, intraperitoneally, by intranasal instillation, byimplantation, by intracavitary or intravesical instillation,intraocularly, intraarterially, intralesionally, transdermally, or byapplication to mucous membranes. The inhibitor may be administered witha pharmaceutically-acceptable carrier. In some embodiments, the thymosinis administered to a subject by injection (e.g., intramuscular,intraarterial, intravascular, intravenous, intraperitoneal, orsubcutaneous). By “pharmaceutically acceptable” is meant a material thatis not biologically or otherwise undesirable, i.e., the material may beincorporated into a pharmaceutical composition administered to a patientwithout causing any significant undesirable biological effects orinteracting in a deleterious manner with any of the other components ofthe composition in which it is contained. When the term“pharmaceutically acceptable” is used to refer to a pharmaceuticalcarrier or excipient, it is implied that the carrier or excipient hasmet the required standards of toxicological and manufacturing testing orthat it is included on the Inactive Ingredient Guide prepared by theU.S. Food and Drug administration.

In some embodiments, a composition of the present invention can beprovided in pharmaceutical compositions comprising a vehicle, such as anartificial membrane vesicle (including a liposome, lipid micelle and thelike), microparticle or microcapsule.

Compositions intended for oral use may be prepared in either solid orfluid unit dosage forms. Fluid unit dosage form can be preparedaccording to procedures known in the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents,flavoring agents, coloring agents and preserving agents in order toprovide pharmaceutically elegant and palatable preparations. An elixiris prepared by using a hydroalcoholic (e.g., ethanol) vehicle withsuitable sweeteners such as sugar and saccharin, together with anaromatic flavoring agent. Suspensions can be prepared with an aqueousvehicle with the aid of a suspending agent such as acacia, tragacanth,methylcellulose and the like.

Solid formulations such as tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients that aresuitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate: granulating anddisintegrating agents for example, corn starch, or alginic acid: bindingagents, for example starch, gelatin or acacia, and lubricating agents,for example magnesium stearate, stearic acid or talc and otherconventional ingredients such as dicalcium phosphate, magnesium aluminumsilicate, calcium sulfate, starch, lactose, methylcellulose, andfunctionally similar materials. The tablets may be uncoated or they maybe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil. Softgelatin capsules are prepared by machine encapsulation of a slurry ofthe compound with an acceptable vegetable oil, light liquid petrolatumor other inert oil.

Aqueous suspensions contain active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxylmethylcellulose, methyl cellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia:dispersing or wetting agents may be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample hepta-decaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl-p-hydroxy benzoate, one or more colouringagents, one or more flavoring agents or one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil, for example peanut oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide palatable oralpreparations. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and colouringagents, may also be present.

Pharmaceutical compositions of the invention may also be in the form ofoil-in-water emulsions. The oil phase may be a vegetable oil, forexample olive oil or peanut oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to known art using those suitable dispersing orwetting agents and suspending agents that have been mentioned above. Thesterile injectable preparation may also be a sterile injectable solutionor a suspension in a non-toxic parentally acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables. Adjuvants such as local anaesthetics,preservatives and buffering agents can also be included in theinjectable solution or suspension.

In some embodiments, the delivery systems suitable include time-release,delayed release, sustained release, or controlled release deliverysystems. In some embodiments, a composition of the present invention canbe delivered in a controlled release system, such as sustained-releasematrices. Non-limiting examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., 1981, J. Biomed. Mater. Res., 15:167-277 andLanger, 1982, Chem. Tech., 12:98-105), or poly(vinylalcohol)],polylactides (U.S. Pat. No. 3,773,919; EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983,Biopolymers, 22:547-556), non-degradable ethylene-vinyl acetate (Langeret al., supra), degradable lactic acid-glycolic acid copolymers such asthe LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). In some embodiments, thecomposition may be administered using intravenous infusion, animplantable osmotic pump, a transdermal patch, liposomes, or other modesof administration. In one embodiment, a pump may be used (see Langer,supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald etal., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574(1989). In another embodiment, polymeric materials can be used. In yetanother embodiment, a controlled release system can be placed inproximity to the therapeutic target, for example liver, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984).Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990). In some embodiments, the composition maybe administered through subcutaneous injection.

In some embodiments, the release of the composition occurs in bursts.Examples of systems in which release occurs in bursts includes, e.g.,systems in which the composition is entrapped in liposomes which areencapsulated in a polymer matrix, the liposomes being sensitive tospecific stimuli, e.g., temperature, pH, light or a degrading enzyme andsystems in which the composition is encapsulated by an ionically-coatedmicrocapsule with a microcapsule core degrading enzyme.

In some embodiments, the release of the composition isgradual/continuous. Examples of systems in which release of theinhibitor is gradual and continuous include, e.g., erosional systems inwhich the composition is contained in a form within a matrix andeffusional systems in which the composition is released at a controlledrate, e.g., through a polymer. Such sustained release systems can bee.g., in the form of pellets, or capsules.

Other embodiments of the compositions administered according to theinvention incorporate particulate forms, protective coatings, proteaseinhibitors or permeation enhancers for various routes of administration,such as parenteral, pulmonary, nasal and oral. Other pharmaceuticalcompositions and methods of preparing pharmaceutical compositions areknown in the art and are described, for example, in “Remington: TheScience and Practice of Pharmacy” (formerly “Remingtons PharmaceuticalSciences”); Gennaro, A., Lippincott, Williams & Wilkins, Philidelphia,Pa. (2000). In some embodiments, the pharmaceutical composition mayfurther include a pharmaceutically acceptable diluent, excipient,carrier, or adjuvant.

The dosage to be administered is not subject to defined limits, but itwill usually be an effective amount, or atherapeutically/pharmaceutically effective amount. The term “effectiveamount” refers to the amount of one or more compounds that renders adesired treatment outcome. An effective amount may be comprised withinone or more doses, i.e., a single dose or multiple doses may be requiredto achieve the desired treatment endpoint. The term“therapeutically/pharmaceutically effective amount” as used herein,refers to the level or amount of one or more agents needed to treat acondition, or reduce or prevent injury or damage, optionally withoutcausing significant negative or adverse side effects. It will usually bethe equivalent, on a molar basis of the pharmacologically active freeform produced from a dosage formulation upon the metabolic release ofthe active free drug to achieve its desired pharmacological andphysiological effects. In some embodiments, the compositions may beformulated in a unit dosage form. The term “unit dosage form” refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient.

In some embodiments, dosing regimen of thymosin includes, without anylimitation, the amount per dose, frequency of dosing, e.g., per day,week, or month, total amount per dosing cycle, dosing interval, dosingvariation, pattern or modification per dosing cycle, maximum accumulateddosing, or warm up dosing, or any combination thereof. In some otherembodiments, dosing regimen of thymosin includes frequency of dosing,e.g., per day or per week.

In yet some embodiments, dosing regimen includes a pre-determined orfixed amount per dose in combination with a frequency of such dose. Forexample, dosing regimen of thymosin includes a fixed amount per dose incombination with the frequency of such dose of thymosin beingadministered to a subject.

In some embodiments, effective amounts of thymosin are amounts which maybe dosage units within a range corresponding to about 0.1-20 mg of TA1,about 1-10 mg of TA1, about 2-10 mg of TA1, about 2-7 mg of TA1, orabout 3-6.5 mg of TA1, and may comprise about 1.6, 3.2 or 6.4 mg of TA1,or about 3.2 or 6.4 mg of TA1. A dosage unit may be administered onceper day, or a plurality of times per day. In some embodiments, TA1 isadministered to a subject at a dosage within a range of about 0.5-10mg/day. In certain embodiments, the TA1 dosage is within a range ofabout 1.5-7 mg/day, or within a range of about 1.6-6.4 mg/day. Incertain embodiments, the TA1 dosage is within a range of about 1.7-10mg/day, about 1.7-7 mg/day, or about 3-7 mg/day. In some embodiments,the effective dosages include about 1.6, 3.2 or 6.4 mg/day.

In some embodiments, the administration provides a serum level ofthymosin at about 0.1 to 1.0 ng/ml. In some embodiments, theadministration provides a peak plasma level after injection of about 100ng/ml. In some embodiments, the half-life of TA1 in the circulation isabout 2 hours.

In certain embodiments, the treatment regimen comprises a plurality ofdays of a pharmaceutical composition comprising TA1, or TA1 can beadministered to the subject during at least a portion of the treatmentregimen.

In certain embodiments, the treatment regimen comprises administeringthe pharmaceutical composition for a period of about 1-10 days, about1-20 days, about 1-30 days, or more.

In certain embodiments, the treatment regimen further comprises about1-5 days, about 5-10 days, about 10-20 days, about 20-30 days or more ofnon-administration of the pharmaceutical composition. In someembodiments, the pharmaceutical composition may be administered daily,once per two days, once per three days, once per four days, once perfive days, once per six days, once per week, for about 1-10 days, about1-20 days, or more, followed by about 1-5 days, about 5-10 days ofnon-administration of the thymosin.

In some embodiments, the methods further comprise monitoring theresponse of the subject after administration to avoid severe and/orfatal immune-mediated adverse reactions due to over-activation andproliferation. In some embodiments, the administration of the immunestimulator is modified, such as reduced, paused or terminated if thepatient shows persistent moderate adverse reactions. In someembodiments, the dosage is modified if the patient fails to respondwithin about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2weeks or more from administration of first dose.

The pharmaceutical composition of the present invention may alsoalleviate, reduce the severity of, or reduce the occurrence of, one ormore of the symptoms associated with melanoma. In some embodiments, suchsymptoms include, but are not limited to, early signs of melanoma arechanges to the shape or color of existing moles or, in the case ofnodular melanoma, the appearance of a new lump anywhere on the skin(such lesions should be referred without delay to a dermatologist). Atlater stages, the mole may itch, ulcerate or bleed. Early signs ofmelanoma include, but not limited to asymmetry in shape, irregularborders, variegated color, greater than 6 mm diameter, and evolving overtime.

In some embodiments, methods of the present invention preventmetastasis, or reduce the rate and/or severity of metastasis.

The present invention also provides a collection of activity profiles ofa panel of biomarkers. As used herein, the term “activity profile”refers to a set of data representing distinctive features orcharacteristics of one or more biomarkers. Such features orcharacteristics include, but are not limited to, transcript abundance,transcript stability, transcription rate, translation rate,post-translation modification, protein abundance, protein stability,and/or protein enzymatic activity, etc. In some embodiments, theactivity profile comprises data related to gene expression level of eachbiomarker. In some embodiments, the collection comprising activityprofiles is obtained from a specific population of subjects. In someembodiments, the specific population of subjects consists of clinicallynormal subjects. In some embodiments, the population consists of patentsresponsive to one or more anti-melanoma agents of the present invention.In some embodiments, the population consists of patients not responsiveto one or more anti-melanoma agents of the present invention.

In some embodiments, the collection comprises activity profiles that arestatistically homogeneous in one or more aspects, e.g., statisticallyhomogeneous in one or more quantitative or semi-quantitative parametersdescribing the features and characteristics of the activity profiles. Insome embodiments, the quantitative parameters include, but are notlimited to, transcript abundance, transcript stability, transcriptionrate, translation rate, post-translation modification, proteinabundance, protein stability, and/or protein enzymatic activity, etc.Whether a group of activity profiles are statistically homogeneous ornot in one or more aspects can be determined by any suitable statistictest and/or algorithm known to one skilled in the art.

In some embodiments, one or more of the biomarkers increase its activityin response to the treatment. In some embodiments, one or more of thebiomarkers decrease its activity in response to the treatment. In someembodiments, one or more of the biomarkers remains its activity inresponse to the treatment. As used herein, the activity of a biomarkercan be a parameter at genomic DNA level, transcriptional level,post-transcriptional level, translational level, post-translationallevel, including, but not limited to gene activity, RNA activity, andprotein activity. The gene activity can be gene copy number, geneamplification number, or promoter activity, etc. RNA activity can bemRNA abundance, synthesis rate, and/or stability, etc. Protein activitycan be protein abundance, synthesis rate, stability, enzymatic activity,phosphorylation rate, modifications, binding activity, etc.

As used herein, when the level of a biomarker goes toward the level of apredetermined standard level, it is called normalization.

As used herein, when the level of a biomarker reduces its speed of goingaway from the level of a predetermined standard level, it is calledstabilization.

In some embodiments, the activity profiles of one or more biomarkers ofthe present invention in a subject are determined and compared to apredetermined standard level. As used herein, the term “predeterminedstandard level” or “predetermined activity profiles” refers tostandardized data or data set representing the average, representativefeatures or characteristics of one or more biomarkers in a specificpopulation. Such features or characteristics include, but are notlimited to, gene copy number, gene amplification, transcript abundance,transcript stability, transcription rate, translation rate,post-translation modification, protein abundance, protein stability,and/or protein enzymatic activity, etc. In some embodiments, thespecific population of subjects are consisting of about 5, about 10,about 20, about 50, about 100, about 200, about 300, about 400, about500, about 1000, about 5000, about 10K, or more individual subjects. Thepredetermined activity profile can be a standardized data or data setcollected before, during, or after the specific population of subjectshas been all exposed to a drug. In some embodiments, the specificpopulation is consisting of subjects responsive to a given drug.

In some embodiments, a subject is “responsive” to a drug for treatingwhen the level of one or more of the biomarkers of the present inventionincreases or decreases toward a pre-determined standard level when thesubject is exposed to a the drug, or when the drug modifies the speed oflevel changes of one or more biomarkers of the present inventioncompared to a placebo. For methods related to detection, quantitationand comparison of biomarker levels, see, e.g., Current Protocols inMolecular Biology, Ed. Ausubel, Frederick M. (2010); Current Protocolsin Protein Science Last, Ed. Coligan, John E., et al. (2010); CurrentProtocols in Nucleic Acid Chemistry, Ed. Egli, Martin (2010); CurrentProtocols in Bioinformatics, Ed. Baxevanis, Andreas D. (2010); andMolecular Cloning: A Laboratory Manual, Third Edition, Sambrook, Joseph(2001), all of which are incorporated herein by reference in theirentirety.

In certain embodiments, when measuring biomarkers or other indicators oftreatment, an “increased” or “decreased” amount or level may include a“statistically significant” amount. A result is typically referred to asstatistically significant if it is unlikely to have occurred by chance.The significance level of a test or result relates traditionally to theamount of evidence required to accept that an event is unlikely to havearisen by chance. In certain cases, statistical significance may bedefined as the probability of making a decision to reject the nullhypothesis when the null hypothesis is actually true (a decision knownas a Type I error, or “false positive determination”). This decision isoften made using the p-value: if the p-value is less than thesignificance level, then the null hypothesis is rejected. The smallerthe p-value, the more significant the result. Bayes factors may also beutilized to determine statistical significance (see, e.g., Goodman S.,Ann Intern Med. 130:1005-13, 1999). In some embodiments, an “increased”or “decreased” amount or level is about 1.1×, 1.2×, 1.3×, 1.4×, 1.5×,2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 25×,30×, 40×, or 50× more or less the amount of a predetermined standard, orthe amount of a determined time point relative to a previous or earliertimepoint.

Also provided are methods for monitoring the efficacy of an active agentin the treatment of cancer. These methods include determining theactivity of one or more biomarkers of the present invention in abiological sample from a patient and providing that informationregarding the biomarkers to an entity that provides a determination orevaluation of the treatment or efficacy based on biomarker information.In some embodiments, the biomarker activity is determined during orafter taking at least one dosage of the active agent of the presentinvention. In some embodiments, the entity can provide a determinationthat treatment with the active agent should be used or should becontinued, if the human subject meets one or more selection criteriadescribed below:

-   -   The human subject has a decreased level of IL-1β activity when        compared to that of the same human subject before the treatment,        and/or has a normalization or stabilization of the IL-1β        activity when compared that of a human subject or a group of        human subjects who are responsive to a treatment of the present        invention;    -   The human subject has an increased level of IL-4 activity when        compared to that of the same human subject before the treatment,        and/or has a normalization or stabilization of the IL-4 activity        when compared that of a human subject or a group of human        subjects who are responsive to a treatment of the present        invention;    -   The human subject has a decreased level of IL-6 activity when        compared to that of the same human subject before the treatment,        and/or has a normalization or stabilization of the IL-1β        activity when compared that of a human subject or a group of        human subjects who are responsive to a treatment of the present        invention;    -   The human subject has a decreased level of IL-10 activity when        compared to that of the same human subject before the treatment,        and/or has a normalization or stabilization of the IL-1β        activity when compared that of a human subject or a group of        human subjects who are responsive to a treatment of the present        invention.

Methods for detecting the levels of nucleic acids, such as RNA or DNAhave been well described and are well known to those of skill in theart. Methods for detecting RNA can include but are not limited toRT-PCR, northern blot analyses, gene expression analyses, microarrayanalyses, gene expression chip analyses, hybridization techniques(including FISH), expression beadchip arrays, and chromatography as wellas any other techniques known in the art. Methods for detecting DNA caninclude but are not limited to PCR, real-time PCR, digital PCR,hybridization (including FISH), microarray analyses, SNP detectionassays, SNP genotyping assays and chromatography as well as any othertechniques known in the art.

Methods for detecting proteins and polypeptides can include but are notlimited to spectrophotometric determination of protein concentration,quantitative amino acid analysis, protein concentration assays,chromatography assays, western blot analyses, gel electrophoresis,(followed by staining procedures including but not limited to CoomassieBlue, Silver stain, Syber Green, Syber Gold), hybridization, multiplexcytokine assays, immunoassays, ELISA, bicinchoninic acid (BCA) proteinassays, Bradford protein assays, and Lowry protein assays as well as anyother techniques known in the art. Protein detection can also includedetecting the levels of stable or active proteins and methods such askinetic assays, kinase assays, enzyme assays and post-translationmodification assays (for example, assays for determining phosphorylationand glycosylation state) can also be employed.

For more methods related to detection, quantitation and comparison ofbiomarker levels, see, e.g., Current Protocols in Molecular Biology, Ed.Ausubel, Frederick M. (2010); Current Protocols in Protein Science Last,Ed. Coligan, John E., et al. (2010); Current Protocols in Nucleic AcidChemistry, Ed. Egli, Martin (2010); Current Protocols in Bioinformatics,Ed. Baxevanis, Andreas D. (2010); and Molecular Cloning: A LaboratoryManual, Third Edition, Sambrook, Joseph (2001), all of which areincorporated herein by reference in their entireties.

In some embodiments, the information regarding the biomarkers isobtained from one or more tests. The test can be performed by thesubject himself/herself, by a doctor, by a nurse, by a test lab, by ahealthcare provider, or any other parties capable of doing the test. Thetest results containing the biomarker information can be then analyzedby the same party or by a second party, such as the subjecthimself/herself, a doctor, a nurse, a test lab, a healthcare provider, aphysician, a clinical trial personnel, a hospital, a lab, a researchinstitute, or any other parties capable of analyzing the test todetermine if the subject is responsive to the drug.

The following examples illustrate various aspects of the invention. Theexamples should, of course, be understood to be merely illustrative ofonly certain embodiments of the invention and not to constitutelimitations upon the scope of the invention.

EXAMPLES Example 1 Treating Melanoma by Thymosin in Subcutaneous B16F10Murine Melanoma Model

B16F10 murine melanoma model is derived from MB16 line by successiveselection of metastatic clones. B16F1 to B16F10 (ATCC Number CRL6475™)were generated, with F10 being passaged in mice for 10 times, and thushighly metastatic. This model is widely used in studying metastasismechanisms, evaluating cancer therapeutics. It is also one of the mostcommon syngeneic models for cancer immunotherapy. Both subcutaneous andexperimental metastasis models are very useful.

To study the effect of thymosin in treating melanoma, thymosin alphapeptide (ZADAXIN) was administered to mice inoculated with B16F10 in twoseparate studies:

TABLE 1 Study No. 1 Design Dose Group Treatment (mg/kg) Notes 1 Vehicle— Vehicle or Ta1 2 TA1 0.2 given bid i.p. for 7 days 3 TA1 2 aftertumors reached 83-96 mm³ 4 TA1 6 Blood & serum collected for biomarkeranalysis at each time point (0, +2, +4 and +7) (n = 12 sacrificed attimepoints 1, +2 and_4 and 24 animals sacrificed at +7 for biomarkeranalysis) All biomarker analysis animals were also measured for tumorsize prior to sacrifice at each timepoint to generate additional tumorsize data Antitumor activity evaluated (n = 6 measured for tumor size ateach timepoint, then sacrificed at end of study). Data from all animalsmeasured or tumor size is depicted in the figures and tables andstatistical analyses.

TABLE 2 Study No. 2 Design Dose Group Treatment (mg/kg) Route Notes 1Vehicle — s.c. Vehicle or Ta1 2 TA1 0.02 s.c. given bid i.p or s.c.depending upon 3 TA1 0.06 s.c. the dose group, for 7 days after 4 TA10.2 i.p. tumors reached 120 mm³ 5 TA1 0.2 s.c. Blood & serum collectedfor 6 TA1 0.6 s.c. biomarker analysis at each time 7 TA1 2 s.c. point (n= 4 animals at 0, +2, +4 8 TA1 6 s.c. and +7) All biomarker analysisanimals were also measured for tumor size prior to sacrifice at eachtimepoint to generate additional tumor size data Antitumor activityevaluated (n = 10 measured for tumor size at each timepoint, thensacrificed at end of study). Data from all animals measured or tumorsize is depicted in the figures and tables and statistical analyses.

Tumor volumes and body weights were monitored throughout the study.Cisplatin was administered to the positive control group in a separatehistorical control study. The negative control group was administeredwith vehicle only in all studies.

B16F10 mice treated with cisplatin typically show reduced tumor volume(FIG. 1A and Table 3). However, these mice also had significantlyreduced body weight as a result of the toxicity of cisplatin (FIG. 1B).

TABLE 3 Tumor size in B16F10 mice treated with cisplatin Tumor Size(mm³) T/C Value T-C (days) Treatment on Day 28 (%) on Day 28 at 800 mm³P value Vehicle 2904 ± 538 — — — Cisplatin  862 ± 233 30 >4 0.006

Animals with B16F10 derived tumor, dosed with ZADAXIN™ (thymalfasin) atall doses tested exhibited reduced tumor growth compared with vehicletreated group (FIG. 2 and FIG. 3).

In study I, The mean tumor size of the vehicle treated group (Group1)reached 1,995 mm³ at day 14 post tumor inoculation. Treatment with TA1at 0.2 mg/kg and 2 mg/kg produced significant antitumor activity at day14 post tumor inoculation. The mean tumor size was 1,148 mm3 (T/Cvalue=57.56%, p value<0.001) and 1,384 mm³ (T/C value=69.36%, pvalue=0.006) with a tumor growth delay of 1.5 and 0.5 day(s)respectively at tumor size of 1,140 mm³. Treatment with TA1 at 6 mg/kgcan delay tumor growth, but the decrease didn't reach a significantdifference (p value=0.146). Moreover, TA1 at 0.2 mg/kg produced betterantitumor activity than TA1 at 6 mg/kg.

In addition, no significant weight changes by group were observed.Therefore, thymosin can be used to treat melanoma.

Example 2 Biomarker Studies

In the studies described above, a group of potential biomarkers weretested in, including IL-7, 11-18, TREM-1, IFN-α, procalcitonin, GM-CSF,IL-1α, IFN-γ, TNF α, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, and IL-1β.The concentration of some particular biomarkers in mice treated byvehicle or ZADAXIN™ (thymalfasin) are shown in FIG. 4A to FIG. 4D. Theresults indicate that these biomarkers can be used to evaluate theefficacy of a cancer treatment (either a single agent or combinationtherapies), to select patients for effective treatment, and to optimizedose and/or regimen for either clinical studies or treatment.

In another study, 8 cytokines (IFNγ, IL-1β, IL-4, IL-5, IL-6, IL-10,IL-12, TNFα) in serum samples of systemic B16F10 murine melanoma modelin C57BL/6 mice with different treatments were analyzed by ELISA. Thetreatments were:

1. Vehicle BID×10 s.c.

2. PD-1 antibody 100 ug/mouse biweek×2 i.p.

3. TA1 0.486 ug/mouse bid×10 s.c.

4. TA1 4.86 ug/mouse bid×10 s.c.

5. TA1 48.6 ug/mouse bid×10 s.c.

6. PD-1 antibody+TA1 (100 ug/mouse+0.486 ug/mouse; biweek×2+bid×10i.p+s.c.)

7. PD-1 antibody+TA1 (100 ug/mouse+4.86 ug/mouse; biweek×2+bid×10i.p+s.c.)

8. PD-1 antibody+TA1 (100 ug/mouse+48.6 ug/mouse; biweek×2+bid×10i.p+s.c.)

9. Cyclophosphomide 300 mg/kg QD×1 i.p.

The concentration of these biomarkers in mice after treatment are shownin FIG. 4E to 4L. The results indicate that these biomarkers could betied to mechanism of action of thymosin, or used as pharmacodynamicmarkers.

Example 3 Treating Melanoma by Thymosin in B16F10 Mouse Lung MetastaticMelanoma Model

Materials and Methods

Mice: Female B6D2F1/Crl mice (Charles River) were 9 weeks old on D1 ofthe study and had a BW range of 19.2 to 24.5 g. The animals were fed adlibitum water (reverse osmosis, 1 ppm Cl) and NIH 31 Modified andIrradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat,and 5.0% crude fiber. The mice were housed on irradiated Enrich-o'Cobs™bedding in static microisolators on a 12-hour light cycle at 20-22° C.(68-72° F.) and 40-60% humidity. DRS-NC specifically complies with therecommendations of the Guide for Care and Use of Laboratory Animals withrespect to restraint, husbandry, surgical procedures, feed and fluidregulation, and veterinary care. The animal care and use program atDRS-NC is accredited by the Association for Assessment and Accreditationof Laboratory Animal Care International, which assures compliance withaccepted standards for the care and use of laboratory animals.

In Vivo Implantation: Test mice were sorted into five treatment groups(n=10), as shown in Table 1. A sixth group of animals was included asthe “look-see” group (n=9). B16-F10 cells were harvested duringlog-phase growth and resuspended at a concentration of 7.5×10⁵ cells/mLin PBS. Each mouse received an intravenous (i.v.) tail vein injection of1.5×10⁵ B16-F10 cells (0.2 mL cell suspension) on D1 of the study.

Test Articles: Anti-PD1 alpha peptide (coded as anti-PD-1-SCE, Lot. No.5177/0214) and thymosin alpha-1 peptide (Thymalfasin, code-named SR1,Lot. No. 1402-224). DRS-NC assigned code names for the purpose ofconfidentiality during in-house testing. Cyclophosphamide (BaxterPharmaceutical, Lot. No. 2E718F, received on Jun. 7, 2013) was includedas a reference control. SR1 was provided as a lyophilized powder (90.7%free base) and was dissolved in PBS to yield a 22.051 mg/mL dosingsolution, which provided a 220.51 mg/kg dosage in a dosing volume of 10mL/kg. Dosing was not adjusted per body weight. Anti-PD-1-SCE antibodywas diluted in PBS to yield a 10.0 mg/mL dosing solution, which provideda 100 mg/kg dosage in a dosing volume of 10 mL/kg. Dosing was notadjusted per body weight. Cyclophosphamide was diluted in saline toyield a 15.0 mg/mL dosing solution, which provided a 300 mg/kg dosage ina dosing volume of 15 mL/kg. Dosing was adjusted per body weight.Cyclophosphamide was prepared once at the beginning of the study andstored at 4° C.

Treatment: Table 4A presents a summary of the treatment plan:

Group 1 animals received PBS s.c. (bid to end) and served as the controltreatment group.

Group 2 received SR1 s.c. at 220.51 mg/kg (200 mg/kg free base) (bid toend).

Group 3 received anti-PD-1-SCE administered i.p. at 100 mg/kg (biwk×3).

Group 4 received both SR1 and anti-PD-1-SCE administered at 220.51 mg/kgs.c. (bid to end) and 100 mg/kg i.p. (biwk×3) respectively.

Group 5 was set as the positive control group, and receivedcyclophosphamide at 300 mg/kg i.p. (qd×1),

Group 6 was set as the “look-see” animals and received no treatment.

Endpoint: The endpoint of the B16MET-e117 study was defined as 100metastases per lung set. Two to three animals from the “look-see” groupwere euthanized in three days intervals beginning on Day 9 and lungmetastatic foci were counted. Total counts were obtained by adding thenumber of foci counted in the superior, middle, inferior, and post-cavallobes of the right lung to the number of foci counted in the left lung.The study was terminated on D16 and all animals were euthanized andtheir metastases counted. Percent inhibition was defined as thedifference between the number of metastatic foci of the designatedcontrol group and the number of metastatic foci of the drug-treatedgroup, expressed as a percentage of the number of metastatic foci of thedesignated control group:% Inhibition=[1−(#Focidrug-treated/#Focicontrol)]×100

Toxicity: Animals were weighed daily for the first five days of thestudy and twice weekly thereafter. The mice were observed frequently forovert signs of any adverse, treatment-related side effects, and clinicalsigns of toxicity were recorded when observed. Acceptable toxicity wasdefined as a group mean body-weight loss of less than 20% during thestudy and not more than one treatment-related (TR) death among tentreated animals. Any dosing regimen resulting in greater toxicity isconsidered above the maximum tolerated dose (MTD). A death is classifiedas TR if attributable to treatment side effects as evidenced by clinicalsigns and/or necropsy, or if due to unknown causes during the dosingperiod or within fourteen days of the last dose. A death is classifiedas non-treatment-related (NTR) if there is no evidence that death wasrelated to treatment side effects.

Statistical and Graphical Analyses: Prism (GraphPad) for Windows 6.02was used for all graphical presentations and statistical analyses. TheMann-Whitney U-test, for analysis of medians, was used to determine thestatistical significance between D16 B16F10 metastatic foci in controland treated groups. Two-tailed statistical analyses were conducted atP=0.05. A “box and whiskers” diagram was constructed to show thedistribution of enumerated metastatic foci for each treatment group onD16.

Prism reports results as non-significant (ns) at P>0.05, significant(symbolized by “*”) at 0.01<P≦0.05, very significant (“**”) at0.001<P≦0.01, and extremely significant (“***”) at P≦0.001. Since theMann-Whitney U-test is a test of significance and does not provide anestimate of the size of the difference between groups, all levels ofsignificance are reported as either significant or non-significant inTable 4B.

Procedures:

-   -   Implant cells (1) day prior to study start.    -   Set up 59 CR female B6D2F1 mice with 1.5×10⁵ B16MET tumor cells        in 0% Matrigel iv    -   tail vein.    -   Cell Injection Volume is 0.2 mL/mouse.    -   Age at Start Date: 8 to 12 weeks.    -   Body Weight: 5/2 then biweek to end    -   Met Count: at endpoint    -   Report any adverse reactions or death to RM, SD, RD or SH        immediately.    -   Any individual animal with a single observation of >than 30%        body weight loss or three consecutive measurements of >25% body        weight loss will be euthanized.    -   Any group with a mean body weight loss of >20% or >10% mortality        will stop dosing.    -   The group is not euthanized and recovery is allowed. Within a        group with >20% weight loss, individuals hitting the individual        body weight loss endpoint will be euthanized. If the group        treatment related body weight loss is recovered to within 10% of        the original weights, dosing may resume at a lower dose or less        frequent dosing schedule. Exceptions to non-treatment body        weight % recovery may be allowed on a case-by-case basis.    -   Endpoint: approximately 100 mets per lung set.    -   Euthanize moribund animals per CRL-NC SOP #687. Animals showing        signs of respiratory distress will also be euthanized as stated        above.

Tumor Cell Culture: B16MET cells were grown to mid-log phase in RPMI1640 medium containing 10% fetal bovine serum, 10 mM HEPES, 2 mMglutamine, 100 units/mL sodium penicillin G, 0.075% sodium bicarbonate,25 μg/mL gentamicin, and 100 μg/mL streptomycin sulfate. The tumor cellswere cultured in tissue culture flasks in a humidified incubator at 37°C., in an atmosphere of 5% CO2 and 95% air.

B16F10 Mouse Lung Metastatic Melanoma Model was developed. About 1.5×10⁵B16-F10 cells (0.2 mL cell suspension) were grown in vitro andadministered intravenously (Day 0) to female B6D2F1/Crl mice. On Day 1dosing with test agents initiated.

A “look see”-untreated-group was set to evaluate, over a period of 15days, the number of metastasis. On day 9, three animals from the “looksee” group were euthanized and lung metastasis were count (ourhistorical data indicates that at this time we would be able to findabout >50 mets in the lung of untreated mice). Three days later otherthree mice were examined. When the met count reached approximately50-100 counts, all groups in the study were sacrificed and final metcounts were established. The “look see” group count was not included inthe final efficacy analysis. 50-100 met count defined as the targetnumber because most of the individual metastasis could be count easilyand mets were less likely to merge with each other difficulty thecounting. See FIG. 5.

In one study (Study I), vehicle (negative control), cyclophosphamide(positive control), TA1, anti-PD-1 or TA1+anti-PD-1 was administered tomice, and met count was evaluated. The result is given in Table 4B belowand in FIG. 6A and FIG. 6B.

TABLE 4A Study 1 Design Treatment Regimen 1 Treatment Regimen 2 Group nAgent Mg/kg Route Schedule Agent Mg/kg Route Schedule 1 10 Vehicle — scbid to end — — — — 2 10 SR1 220.51^(a) sc bid to end — — — — 3 10Anti-PD-1-SCE 100^(a)   ip biwk x 2 — — — — 4 10 SR1 220.51^(a) sc bidto end Anti-PD-1-SCE 100^(a) ip biwk x 2 5 10 Cyclophosphamide 300    ipqd x 1 — — — — 6 9 LOOK SEE — iv qd x 1 — — — — ^(a)μg/animal Vehicle =PBS

TABLE 4B Study 1 Result Mean Statistical Mean Treatment Regimen MetPercent Significance BW Deaths Group n Agent mg/kg Route Schedule CountSEM n Inhibition Vs G1 Vs G4 Nadir TR NTR 1 10 Vehicle — sc bid to end105.9 9.8 10 — — — −2.1% 0 0 Day 4 2 10 SR1 220.51^(a) sc bid to end72.5 9.6 10 31.5 * ns — 0 0 3 10 Anti-PD-1-SCE 100^(a)   ip biwk x 358.7 7.2 10 44.6 *** ns — 0 0 4 10 SR1 220.51^(a) sc bid to end 58 5.410 45.2 *** — — 0 0 Anti-PD-1-SCE 100^(a)   ip biwk x 3 5 10Cyclophosphamide 300    ip Qd x 1 0.5 0.4 10 99.5 *** — −4.3% 0 0 Day 3^(a)μg/animal Days in Progress = 16 n = number of animals in a group notdead from accidental causes (NTR deaths excluded from TGD calculations)Percent Inhibition = [1 − (T/C)] × 100, compared to Group 1 StatisticalSignificance (ANOVA-Dunnett or Student's t -test): ne = not evaluable,ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001,compared to group indicated Mean BW Nadir = lowest group mean bodyweiaht, as % change from Day 1; — indicates no decrease in mean bodyweight was observed TR = treatment-related death; NTR =non-treatment-related death

The percent group mean body weigh changes from Day 1 in B16MET-e117 miceare shown in FIG. 6B.

The result indicates that thymosin decreases metastases to lung inmelanoma model, but there is no additive effect with anti-PD-1 at theseparticular doses.

In a second study (Study II), vehicle (negative control),cyclophosphamide (positive control), TA1, anti-PD-1 or TA1+anti-PD-1 wasadministered to mice at different doses. The study design and result aregiven in Table 5A and Table 5B below and in FIG. 7A to FIG. 7C.

Treatment Plan for Study II: In this study, thirteen groups of femaleB6D2F1 mice were dosed in accordance with the protocol in Table 5A. TheSR1 and the vehicle were each administered subcutaneously (s.c.) twicedaily for the duration of the study (bid to end). The anti-PD1 antibodywas administered intraperitoneally (i.p.) twice a week for two weeks(biwk×2). A single dose of cyclophosphamide was administered i.p.(qd×1). Table 5A presents a summary of the treatment plan.

Group 1 animals received PBS and served as the control treatment group.

Groups 2-4 received SR1 at 0.4862, 4.862 and 48.62 μg/mouse (0.441, 4.41and 44.1 μg/mouse free base), respectively.

Groups 5 and 6 received anti-PD-1 administered at 33.33 and 100μg/mouse, respectively.

Groups 7-9 received SR1 at 0.4862, 4.862 and 48.62 μg/mouse incombination with anti-PD-1 administered at 33 μg/mouse, respectively

Groups 10-12 received SR1 at 0.4862, 4.862 and 48.62 μg/mouse incombination with anti-PD-1 administered at 100 μg/mouse, respectively

Group 13 was set as the positive control group, and receivedcyclophosphamide at 300 mg/kg.

Group 14 was set as the “look-see” animals and received no treatment.

TABLE 5A Study II Design Regimen 1 Regimen 2 Group n Agent μg/animalRoute Schedule Agent Mg/kg Route Schedule  1# 10 Vehicle (PBS) — sc bidto end — — — — 2 10 SR1   0.4862 sc bid to end — — — — 3 10 SR1  4.86 scbid to end — — — — 4 10 SR1 48.62 sc bid to end — — — — 5 10Anti-PD-1-SCE 33.33 ip biwk x 2 — — — — 6 10 Anti-PD-1-SCE 100    ipbiwk x 2 — — — — 7 10 SR1   0.4862 sc bid to end Anti-PD-1-SCE 33.33 ipbiwk x 2 8 10 SR1  4.86 sc bid to end Anti-PD-1-SCE 33.33 ip biwk x 2 910 SR1 48.62 sc bid to end Anti-PD-1-SCE 33.33 ip biwk x 2 10  10 SR1  0.4862 sc bid to end Anti-PD-1-SCE 100 ip biwk x 2 11  10 SR1  4.86 scbid to end Anti-PD-1-SCE 100 ip biwk x 2 12  10 SR1 48.62 sc bid to endAnti-PD-1-SCE 100 ip biwk x 2 13  10 Cyclophosphamide 300*   ip qd x 1 —— — — 14  8 LOOK SEE — iv qd x 1 — — — — #Control Group *Mg/kg

TABLE 5B Study II Result Treatment Regimen Mean Inhibi- StatisticalSignificance Mean μg/ Sched- Met tion vs vs vs vs vs vs BW Deaths Groupn Agent animal Route ule Count SEM n % G1 G2 G3 G4 G5 G6 Nadir TR NTR 110 Vehicle — sc bid to end 160 16.2 10 — — — — — — — — 0 0 2 10 SR1  0.4862 sc bid to end 186 12.6 10 −17%  ns — — — — — — 3 10 SR1  4.86sc bid to end 182 22.9 10 −14%  ns — — — — — — 0 0 4 10 SR1 48.62 sc bidto end 167 27.1 10 −5% ns — — — — — — 0 0 5 10 Anti-PD-1-SCE 33.33 ipbiwk x 2 131 12.4 10 18% ns — — — — — −0.9% 0 0 Day 2 6 10 Anti-PD-1-SCE100    ip biwk x 2 127 11.4 10 21% ns — — — — — −0.9% 0 0 Day 2 7 10 SR1  0.4862 sc bid to end 102 15.4 10 36% * *** — — ns — −1.4% 0 0Anti-PD-1-SCE 33.33 ip biwk x 2 Day 2 8 10 SR1  4.86 sc bid to end 14411.4 10 10% ns — ns — ns — −1.5% 0 0 Anti-PD-1-SCE 33.33 ip biwk x 2 Day2 9 10 SR1 48.62 sc bid to end 103 18.3 10 36% ns — — ns ns — — 0 0Anti-PD-1-SCE 33.33 ip biwk x 2 10 10 SR1   0.4862 sc bid to end 10316.2 10 36% ns *** — — — ns −1.0% 0 0 Anti-PD-1-SCE 100    ip biwk x 2Day 2 11 10 SR1  4.86 sc bid to end 96 10.9 10 40% * — ** — — ns — 0 0Anti-PD-1-SCE 100    ip biwk x 2 12 10 SR1 48.62 sc bid to end 98 9.6 1039% * — — * — ns −0.8% 0 0 Anti-PD-1-SCE 100    ip biwk x 2 Day 2 13 10Cyclophos- 300*   ip qd x 1 2 0.5 10 99% *** — — — — −8.0% 0 0 phamideDay 2 *mg/kg Days in Progress = 15 n = number of animals in a group notdead from accidental causes (NTR deaths excluded from TGD calculations)Percent Inhibition = [1 − (T/C)] × 100, compared to Group 1 Mean BWNadir = lowest group mean body weight, as % change from Day 1; —indicates no decrease in mean body weight was observed TR =treatment-related death; NTR = non-treatment-related death StatisticalSignificance (Anova Dunnett's test for Group 1, 2, 5, 7; Group 1, 3, 5,8; Group 1, 4, 5, 9; Group 1, 2, 6, 10; Group 1, 3, 6, 11;Kruskal-Wallis-Dunn test for Group 1, 4, 6, 12 or Unpaired t-test withWelch's correction for G1 vs. G13): ns = not significant, * = P < 0.05,** = P < 0.01, *** = P < 0.001, compared to group indicated

The result indicates that at certain doses the combination of ZDX(Ta1)+anti-PD-1 treated groups exhibited fewer metastases compared togroups treated with Ta1 or anti-PD-1 alone. Such result supports thatthymosin treatment can provide positive statistically significantreduction in metastases in B16F10 murine lung metastases model, withefficacy trends in combination with an anti-PD-1 antibody.

The activity of 44 biomarkers in mice under each treatment was analyzed.Among these biomarkers, several biomarkers showed statisticallysignificant differential activity between mice treated only with Ta1 oranti-PD-1 alone, and mice treated with a combination of Ta1 andanti-PD-1. Such biomarkers include, but are not limited toApolipoprotein A-I, Leptin, Lymphotactin, Macrophage Colony-StimulatingFactor-1 (M-CSF-1), Monocyte Chemotactic Protein-5 (MCP-5), Stem CellFactor (SCF), and Vascular Cell Adhesion Molecule-1 (VCAM-1).

A similar study was performed in an independent laboratory to confirmthe results. The result is shown in Table 6 and FIG. 8. Interestingly,in this study, the anti-PD-1 did not work as well as the study above,but the TA1 did. This can be attributed to lot differences in theanti-PD-1 antibody sourced by both labs and to the fact that B16F10doesn't always respond to anti-PD-1 in lung met models. The importantthing is that the combination of anti-PD-1 and TA1, especially at thelow dose of TA1 showed a statistically significant decrease in lung metsand there were positive trends in the other two combination groups.Thus, these preliminary studies do confirm additive or possiblysynergistic effects of the two in combination, especially when one ofthe two doesn't seem to work as single agent.

TABLE 6 Antitumor Activity of Thymalfasin as Single Agent and inCombination with PD-1 Antibody in the Treatment of Systemic B16F10Murine Melanoma Model Number of metastasis foci on day 13 InhibitionGroup Treatment (Mean ± SEM) (%) P value* 1 Vehicle 115 ± 18 — — 2 PD-1antibody 121 ± 12 −4.87 0.684 (100 μg/mouse) 3 TA1 (0.486 μg/mouse) 92 ±9 20.35 0.288 4 TA1 (4.86 μg/mouse)  67 ± 12 41.83 0.003 5 TA1 (48.6μg/mouse)  66 ± 10 42.26 0.008 6 PD-1 antibody 60 ± 8 47.83 0.002 (100μg/mouse) + TA1 (0.486 μg/mouse) 7 PD-1 antibody 90 ± 9 21.91 0.248 (100μg/mouse) + TA1 (4.86 μg/mouse) 8 PD-1 antibody 78 ± 9 32.61 0.06 (100μg/mouse) + TA1 (48.6 μg/mouse) 9 Cyclophosphomide  0 ± 0 100 <0.001(300 mg/kg)

Unless defined otherwise, all technical and scientific terms herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materials,similar or equivalent to those described herein, can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. All publications, patents, and patentpublications cited are incorporated by reference herein in theirentirety for all purposes.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

What is claimed:
 1. A method of treating cancer or a metastasis thereofin a subject comprising administering a composition comprisingtherapeutically effective amount of a first immune stimulator and asecond immune stimulator, wherein the first immune stimulator is analpha thymosin peptide, and the second immune stimulator is a programmedcell death-1 (PD-1) inhibitor.
 2. The method of claim 1, wherein thesubject is a human.
 3. The method of claim 1, wherein the second immunestimulator is administered to said subject at a dosage of about0.01-1000 mg/day.
 4. The method of claim 1, wherein the alpha thymosinpeptide is administered to the subject during at least a portion of thetreatment at a dosage within a range of about 0.5-10 mg/day.
 5. Themethod of claim 4, wherein said dosage of the alpha thymosin peptide iswithin a range of about 0.1-10 mg/day.
 6. The method of claim 1, whereinthe alpha thymosin peptide is thymosin alpha 1 (TA1).
 7. The method ofclaim 6, comprising administration of TA1 daily for a period of about1-10 days, followed by about 1-5 days of non-administration of TA1. 8.The method of claim 7, wherein TA1 is administered daily for about 3-5days, followed by about 2-4 days of non-administration of TA1.
 9. Themethod of claim 8, wherein TA1 is administered daily for about 4 days,followed by about 3 days non-administration of TA1.
 10. The method ofclaim 1, wherein the combination further comprises a kinase inhibitor.11. The method of claim 10, wherein said kinase inhibitor comprisessorafenib.
 12. The method of claim 10, wherein said kinase inhibitor isadministered to said patient at a dosage within a range of about 10-200mg/day.
 13. The method of claim 1, wherein the composition furthercomprises an antineoplastic heat shock apoptosis activator (HSAA). 14.The method of claim 13, wherein said HSAA comprises STA-4783(elesclomol).
 15. The method of claim 13, wherein said HSAA isadministered to said patient at a dosage within a range of about0.01-100 mg/kg/day.
 16. The method of claim 1, wherein said combinationfurther includes administration of an antibody against cytotoxic Tlymphocyte-associated antigen 4 (CTLA4).
 17. The method of claim 16,wherein said CTLA4 antibody comprises 9H10, MDC010, 1F4, BNI3, Q01, A01,M08, 1B8, WKH203, ab9984, ab13486, ipilimumab, ticilimumab or acombination thereof.
 18. The method of claim 16, wherein said CTLA4antibody is administered to said patient at a dosage within a range ofabout 0.001-50 mg/kg/day.
 19. The method of claim 1, wherein saidcombination further includes administration of an alkylatingantineoplastic agent (AlkAA).
 20. The method of claim 19, wherein thealkylating antineoplastic agent (AlkAA) comprises dacarbazine (DTIC).21. The method of claim 19, wherein the alkylating antineoplastic agent(AlkAA) is administered to said patient at a dosage within a range ofabout 700-1300 mg/kg/day.
 22. The method of claim 1, wherein the methodfurther comprising administering a chemotherapeutic agent to thesubject.
 23. The method of claim 22, wherein the chemotherapeutic agentis dacarbazine (DTIC) or cisplatin.
 24. The method of claim 1, whereinthe cancer is melanoma.
 25. The method of claim 24, wherein thecomposition further comprises an additional anti-melanoma agent.